Drug delivery system

ABSTRACT

The invention relates to a time controlled, immediate release drug delivery system for oral administration of a first active ingredient to a subject in need thereof. The invention additionally relates to a dual drug delivery device, comprising the time controlled, immediate release drug delivery system according to the invention, further comprising a second coating comprising a second active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 15/687,303,filed Aug. 25, 2017, which is a Division of application Ser. No.14/117,619, having an international filing date of 14 May 2012, now U.S.Pat. No. 9,763,884, issued Sep. 19, 2017, which is the national phase ofPCT application PCT/NL2012/050336 having an international filing date of14 May 2012, which claims benefit of European application Nos.11166091.6, filed 13 May 2011, 11181165.9, filed 13 Sep. 2011; and11183732.4, filed 3 Oct. 2011. The contents of the above patentapplications are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

The invention relates to the field of drug formulation and drugdelivery. More specifically, the invention relates to a time controlled,immediate release drug delivery system. The invention additionallyrelates to a dual drug delivery device comprising the time controlled,immediate release drug delivery system for the time controlled,immediate release of a first active ingredient and controlled release ofa second active ingredient. The invention further relates to aformulation for the sublingual administration of an active ingredient.

Pharmaceutical research is increasingly focusing on smart drug deliverysystems that improve desirable therapeutic objectives while minimizingside effects. The present invention provides smart drug delivery systemsfor designing drug formulations that allow controlled release, such astimed release formulations, including oral formulations.

The art shows various solutions to the problem of controlled release ofan active ingredient. For example, diclofenac is poorly soluble inacidic medium, affecting the solubility and absorption of the drug. Adelayed release mechanism formulation, also termed enteric coatingsystem, prevents release of the drug in the acidic environment of thestomach and allows release in the more favorable environment of thesmall intestine. Various materials, e. g., cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate,and acrylic polymers, have been used as gastroresistant, enterosolublecoatings for delayed drug release in the intestine (Xu and Lee, Pharm.Res. 10 (8), 1144-1152 (1993)). Enteric coating systems, which aresoluble at higher pH values, are frequently used for late intestinal andcolon-specific delivery systems.

WO97/25979 describes a drug-delivery system for targeting various partsof the gastrointestinal tract. A core containing a drug is coated with ahydrophobic polymer which contains hydrophilic, non-water-solubleparticles embedded therein. These particles serve as channels foraqueous medium entering the core and for the release of drugs bydiffusion through these channels.

A further example of a delayed drug delivery system is provided byWO99/018938. WO99/018938 describes a gastrointestinal delivery systemcomprising a drug in combination with a swellable core material. Thecore is surrounded by a water-insoluble coating material comprisingparticulate water-insoluble material. Upon exposure to aqueous liquid,the particulate matter takes up liquid and forms channels in the coatthat allow entry of aqueous liquid to the core. The inner coat burstswhen the core is swollen thereby releasing the drug from the deliverysystem.

Dual drug delivery devices are designed to release a drug at 2 differentrates or in 2 different periods of time, or to release two or moredifferent drugs at different periods of time in different compartments.Dual drug delivery devices control the release rate of one or more drugsto maximize the therapeutic effect of these drugs. Suitable candidatedrugs for a dual phase mode of administration include nonsteroidalanti-inflammatory drugs (NSAIDs) and antihypertensive, antihistaminic,and anti-allergic ingredients. In a first phase, the drug is quicklyreleased to provide maximum relief within a short time frame. This isfollowed by a sustained release phase to avoid a need for repeatedfrequent administration.

Suitable devices for use as a biphasic release system are compresseddouble-layer tablets and “core-within-coating” systems, which involvesthe use of a sustained release tablet as a compressed core which iscoated over the whole surface with a disintegrating formulation. Boththe core tablet and the outer coating contain a drug.

Some biphasic release devices exist in the art. WO93/009771 describes atwo pulse tablet of flutamide for the treatment of prostate cancer. Thefirst pulse is obtained from an immediate release layer while the secondpulse is obtained from a core which contains a solid dispersion of theflutamide in a carrier. The immediate release layer and the core areseparated by a film layer of an enteric coating.

Multiparticulates also provide a biphasic release system. WO94/12160describes a capsule which contains a plurality of pellets with varyingdelay times to drug release. By mixing pellets of different delay timesone can obtain pulsatile delivery of the drug. The drug is contained inthe pellet along with an osmotic ingredient. The pellets are coated witha water permeable, water-insoluble film that allows water diffusion intothe pellet. The osmotic ingredient dissolves in the water causing thepellet to swell and eventually burst to release drug. The osmoticingredient that is contained in a pellet, and the coating of pellet, aretwo of the variables that determine the delay time of a drug that iscontained in a pellet.

WO 98/51287 describes a pulsatile system based on multiple particles ina dosage form. The drug release from the particle is controlled bycombinations of controlled release layers, swelling layers and coatinglayers. The controlled release layer is a crosslinked poly (acrylicacid) polymer of high molecular weight admixed with a water solublepolymer.

A further biphasic drug delivery device is provided by WO00/074655,which system is based on the drug delivery system provided inWO97/25979. The inner coat of the drug delivery system is additionallysurrounded by an outer coat that contains additional amounts of adesired ingredient. When the delivery device enters the gastrointestinaltract, the outer coat releases the desired ingredient contained thereinand disintegrates, exposing the inner coat. By controlling parameters inthe device, such as the core material, carrier material in the coating,and particulate matter, the location of release of both drug pulses canbe controlled.

The afore mentioned drug delivery systems, while effective in delayingrelease of a drug to specific parts of the gastrointestinal tract suchas, for example, the small intestine or the colon, were found to beineffective in providing a drug in a short pulse after a certain periodof time, irrespective of the presence in a specific body compartment.

There is a clear need for a drug delivery system that releases a drugafter a predetermined period of time (a lag time) followingadministration of the drug delivery system. In addition, there is a needfor a drug delivery device that combines a drug delivery system that iseffective in delivering a drug in a short pulse after a predeterminedperiod of time with a drug delivery system that provides immediaterelease of a drug at an earlier point in time after administration,preferably in the oral cavity.

Therefore, the invention provides a time controlled, immediate releasedrug delivery system for oral administration of a therapeuticallyeffective amount of a first active ingredient to a subject in needthereof, comprising a disintegrating core comprising cellulose, a fillerselected from an organic and/or an inorganic salt, and a first activeingredient, said system further comprising a first coating surroundingthe core, said first coating comprising an outer surface, said firstcoating further comprising a hydrophobic polymer and a water-solubleand/or water-insoluble hydrophilic substance.

A core according to the invention comprises

a first active ingredient in a relative amount of preferably between 0.1and 60% (w/w; based on the total weight of the core), more preferredbetween 0.1 and 30% (w/w; based on the total weight of the core), morepreferred between 5 and 25% (w/w based on the total weight of the core),

cellulose in a relative amount of preferably between 10 and 60% (w/wbased on the total weight of the core), more preferred between 10 and50% (w/w based on the total weight of the core (w/w based on the totalweight of the core), and

a filler selected from an organic and/or inorganic salt in a relativeamount of preferably between 10 and 70% (w/w based on the total weightof the core), more preferred in an amount of between 10 and 60% (w/wbased on the total weight of the core).

Throughout this specification, the term “comprising” and its grammaticalequivalents indicate that the components listed are present and thatother components may be present or not. The term “comprising” preferablyhas the meaning of “consisting only of”.

The core is preferably pressed or compacted into a solid. A preferredcore is a tablet. The term “tablet” encompasses a “capsule” and a“caplet”. The preferred size of the core of a drug delivery systemaccording to the invention ranges from a few millimeters to about onecentimeter. Further excipients may include diluents, binders orgranulating ingredients, a carbohydrate such as starch, a starchderivative such as starch acetate and/or maltodextrin, a polyol such asxylitol, sorbitol and/or mannitol, lactose such as α-lactosemonohydrate, anhydrous α-lactose, anhydrous β-lactose, spray-driedlactose, and/or agglomerated lactose, a sugar such as dextrose, maltose,dextrate and/or inulin, or combinations thereof, glidants (flow aids)and lubricants to ensure efficient tabletting, and sweeteners orflavours to enhance taste.

Said first active ingredient can be a single active ingredient, or amixture of two or more active ingredients. It is preferred that each ofthe active ingredients in a mixture of active ingredients is present ina relative amount of between 0.1 and 30% (w/w), more preferred between 5and 25% (w/w),

A preferred time controlled, immediate release drug delivery systemaccording to the invention comprises an immediate release formulationcomprising a compressed core containing one or more active ingredientssurrounded with a coating, wherein release of the active ingredient fromthe core is caused by rupture of the coating after a pre-definedlag-time. Preferably, the core disintegrates immediately after ruptureor dissolution of the coating.

The term cellulose comprises powdered cellulose, agglomerated cellulose,microcrystalline cellulose and/or combinations thereof. The termcellulose includes purified cellulose, methylcellulose, hydroxypropylmethylcellulose, and carboxy methyl cellulose. Powdered cellulose iscomposed mainly of cellulose obtained by decomposing pulp.Microcrystalline cellulose comprises a special grade of alpha cellulose.

A preferred cellulose is microcrystalline cellulose. A preferredmicrocrystalline cellulose has a nominal particle size of between 30 and250 m, preferably of between 50 and 180 m. A further preferredmicrocrystalline cellulose comprises a moisture of between 0.1 and 7.5%,more preferred between 1 and 5.0%. A preferred microcrystallinecellulose is selected from microcrystalline cellulose with a nominalparticle size of 50 m and a moisture of 3.0 to 5.0% such as, forexample, Avicel PH 101; a microcrystalline cellulose with a nominalparticle size of 100 m and a moisture of 3.0 to 5.0% such as, forexample, Avicel PH 102; and a microcrystalline cellulose with a nominalparticle size of 180 m and a moisture less than 1.5% such as, forexample, Avicel PH 200. The amount of said microcrystalline cellulose ispreferably more than 10% (w/w; based on the total weight of the core),more preferred more than 20% (w/w), more preferred more than 30%, mostpreferred more than about 35%. It is further preferred that the amountof microcrystalline cellulose is less than 60%, more preferred less than50%, more preferred less than 45% (w/w, based on the total weight of thecore).

A preferred core according to the invention comprises a filler. Saidfiller is preferably present in an amount of between 10 and 70% (w/w;based on the total weight of the core), more preferred between 20% and60% (w/w), more preferred between 30% and 50% (w/w), such as, forexample, 35% (w/w). Said filler is selected from the group of an organicsalt and an inorganic salt. An organic salt is preferably selected fromcalcium citrate, magnesium citrate, calcium lactate, sodium lactate,magnesium lactate, calcium fumarate and magnesium fumarate. A mostpreferred filled is an inorganic salt. An inorganic salt according tothe invention is preferably selected from calcium sulphate dehydrate,calcium silicate, silicium phosphate, calcium carbonate, anhydrousdibasic calcium phosphate, dibasic calcium phosphate monohydrate,tribasic calcium phosphate, sodium phosphate, sodium chloride, potassiumphosphate, potassium sulphate, potassium chloride, sodium carbonate,magnesium carbonate, and magnesium oxide. The total amount of a solublefiller such as sodium lactate and sodium chloride is preferably below50% (w/w; based on the total weight of the core). The selection of afiller is further determined by the intrinsic stability of the activeingredient in the core in combination with a filler or combination offillers, as is known to the person skilled in the art. The core mayfurther comprise a lubricant such as magnesium stearate, talc and thelike. A preferred core comprises anhydrous dibasic calcium phosphate andmagnesium stearate. The amount of said anhydrous dibasic calciumphosphate is preferably more than 10% (w/w; based on the total weight ofthe core), more preferred more than 20% (w/w), more preferred more than30%, most preferred more than about 35%. It is further preferred thatthe amount of anhydrous dibasic calcium phosphate is less than 70%, morepreferred less than 60%, more preferred less than 50%, more preferredless than 45% (w/w, based on the total weight of the core). The amountof magnesium stearate is preferably between 0.1% (w/w; based on thetotal weight of the core) and 10% (w/w), more preferred between 0.5 and5% (w/w).

The core additionally may comprise one or more disintegrants that, as apure material, form a gel upon exposure to an aqueous liquid. Apreferred disintegrant comprises one of more of a water-insoluble,gel-forming disintegrant. When present, said disintegrant such as awater-insoluble, gel-forming disintegrant is preferably present in arelative amount of between 0.5 and 20% (w/w). Disintegrants aresubstances or a mixture of substances that facilitate the breakup ordisintegration of a tablet. Break up of a tablet results in smallerparticles of which the ingredients, including the first activeingredient, are more rapidly available for uptake, compared to a wholetablet. Drug dissolution can be improved significantly with the additionof disintegrating ingredients into the formulation. Preferreddisintegrants induce disintegration of a tablet by wicking, deformation,and/or the induction of electric repulsive forces between particles.

A preferred disintegrant according to the invention is selected fromsodium starch glycolate (Primojel®), cross-linked sodium carboxymethylcellulose, for example ACDISOL®, cross-linked polyvinylpyrrolidone(Crospovidone) and low-substituted hydroxypropylcellulose (L-HPC) havinga hydroxypropoxyl content in the range of 5.0 to 16.0% by weight and anapparent average degree of polymerization in the range of 350 to 700.Said L-HPC preferably has a low particle size, preferably below 10microns average particle size, more preferred below 5 micron, such as,for example, LH41. Said water-insoluble, gel-forming disintegrant ispreferably present in a relative amount of between 0.0 and 6% (w/w). Theamount of said water-insoluble gel-forming disintegrant is preferablyless than 6% (w/w; based on the total weight of the core), morepreferred less than 5% (w/w), most preferred less than 4%.

A preferred composition of a core according to the invention comprises afirst active ingredient, a microcrystalline cellulose, for examplePHARMACEL® pH102 or PHARMACEL® pH200, anhydrous dicalcium phosphate, acrosslinked sodium carboxy methylcellulose, for example croscarmellose,and magnesium stearate. Microcrystalline cellulose and crosslinkedsodium carboxy methylcellulose are preferably present in a ratio ofbetween about 6:1 (w/w) to 14:1 (w/w), preferably between 7.5 (w/w) and12.5 (w/w). Preferred ratios are about 10:1 (w/w) and about 8:1 (w/w).An effect of such ratio is that the core, while gel-forming, does notsubstantially swell prior to disintegration. A preferred ratio ofanhydrous dibasic calcium phosphate and microcrystalline cellulose isbetween 3:1 (w/w) and 1:3 (w/w), more preferred between 2:1 (w/w) and1:2 (w/w), most preferred in about 1:1 (w/w).

The total weight of a core according to the invention is preferablybetween 50 and 500 milligram, more preferred between 200 and 400milligram, more preferred between 300 and 400 milligram, such as about340 milligram.

A core according to the invention is surrounded by a first coating, saidfirst coating comprising an outer surface, said first coating furthercomprising a hydrophobic polymer and a (water-soluble and/orwater-insoluble) hydrophilic substance. The first coating preferablydoes not comprise a drug. When present, a plasticizer such as, forexample, dibutyl phthalate, triethyl citrate, acetyl triethyl citrate,dibutyl sebacate, diethyl phthalate, triacetin and/or tributyl citrateis preferably present in an amount of at most 0.5% (w/w; based on thetotal weight of the time controlled, immediate release drug deliverysystem). The first coating preferably does not comprise a plasticizer.

The first coating is preferably sprayed, for example with a nozzle, ontothe core. For this, the hydrophobic polymer and water-soluble and/orwater-insoluble hydrophilic substance are suspended or dissolved, forexample in water or an organic solvent or a mixture thereof, and sprayedonto the core until a predetermined average thickness of the firstcoating is obtained. A preferred organic solvent is an alcohol, forexample ethanol. The amount of the first coating is preferably betweenabout 0.5 and 30% (w/w) of the total weight of the time controlled,immediate release drug delivery system, more preferred between about 1and 20% (w/w).

A hydrophobic coating polymer according to the invention is preferablyselected from water-insoluble coating materials such as cellulosederivates and polymethacrylates that are generated, for example, bycopolymerization of methacrylate monomers with hydrophobic groups.Preferred polymethacrylate hydrophobic polymers are EUDRAGIT® RL,EUDRAGIT® RS, EUDRAGIT® NE, and EUDRAGIT® S.

Preferred cellulose derivates are selected from ethylcellulose andderivatives thereof. A most preferred hydrophobic polymer of the firstcoating of a drug delivery system according to the invention comprisesethylcellulose. Ethylcellulose forms a mechanically weak hydrophobicfilm that ruptures easily. The core contains a drug in combination witha water-insoluble, gel-forming disintegrant that disintegrates uponcontact with an aqueous medium. The formation of pores in thehydrophobic film, and the influx of water into the core, causes therupture of the ethylcellulose coating. When the coating is ruptured, thecore disintegrates within minutes followed by the release of the drug. Apreferred ethylcellulose is ETHOCEL®.

A hydrophilic substance according to the invention preferably is awater-insoluble hydrophylic substance, preferably a water-insolublehydrophylic polymer. It is further preferred that said first coatingcomprises pores prior to exposure to an aqueous liquid. The poresfunction as channels that interconnect the core with the outer surfaceof the inner coat for controlling the entry of aqueous liquid into thecore. Said pores are present, for example, when the water-insolublehydrophilic substance is or comprises a water-insoluble hydrophylicpolymer, preferably cellulose. Preferred celluloses are cellulosederivatives such as, for example, hydroxypropylcellulose, crosslinkedhydroxyethylcellulose, crosslinked hydroxypropylmethylcellulose andmicrocrystalline cellulose. Cellulose formed channels that connect thedrug-containing core with the outside of the tablet. The cellulosethereby controls the rate at which water is being transported throughthe channels into the core. When sufficient water reaches the core, thecore looses its structural integrity. The core will disintegrate,followed by rupture of the coating and release of the drug. A preferredcellulose is a microcrystalline cellulose with a nominal particle sizeof between 20 and 200 micron and a moisture of less than 5%. A preferredmicrocrystalline cellulose comprises a microcrystalline cellulose with anominal particle size of about 150 micron and a moisture of 3.0 to 5.0%such as, for example, Avicel® PH-102 SCG; a microcrystalline cellulosewith a nominal particle size of about 100 micron and a moisture lessthan 5.0% such as, for example Avicel® HFE-102; a microcrystallinecellulose with a nominal particle size of about 20 micron and a moistureless than 5.0% such as, for example, Avicel® PH-105. Further preferredwater insoluble hydrophilic substances include dicalcium phosphate.

An advantage of using smaller particles of less than 50 micron, e.g.Avicel® PH-105, is that the coating suspension has better flowproperties, which improves the overall film coating process. A preferredfirst coating comprises Ethocel® and Avicel PH-105 as a water-insolublehydrophylic substance. Preferred mass ratios of a hydrophobic coatingpolymer such as Ethocel® and a water-insoluble hydrophilic substancesuch as Avicel are between 1:5 and 5:1, more preferred between 1:4 and3:1, more preferred between 1:3 and 2:1, most preferred about 1:2.

In another embodiment, a hydrophilic substance according to theinvention preferably is a water-soluble hydrophylic substance. Thiscoating preferably does not comprise pores or only a few pores prior toexposure to an aqueous liquid. It is preferred that the water-solublehydrophilic substance forms pores in the hydrophobic polymer uponexposure to an aqueous liquid. A preferred water-soluble hydrophilicsubstance comprises lactose, mannitol and/or sodium chloride. Apreferred lactose is PHARMATOSE®.

A preferred first coating comprises Ethocel® and lactose as awater-soluble hydrophylic substance. Preferred mass ratios of ahydrophobic coating polymer such as Ethocel® and a water-solublehydrophilic substance such as lactose are between 1:5 and 5:1, morepreferred between 1:3 and 3:1, more preferred between 1:2 and 2:1, mostpreferred about 1:1.

The relative amount of a first coating is preferably between 4 and 20%(w/w; based on the total weight of the drug delivery system), morepreferred between 8 and 15% (w/w), most preferred about 12% (w/w).Therefore, a preferred first coating has a weight of between 10 and 75milligram, more preferred between 25 and 50 milligram, most preferredabout 40 milligram.

A time controlled, immediate release drug delivery system according tothe invention allows control of the release of a first active ingredientafter hydration of the drug delivery system. Said time controlled,immediate release is essentially independent of pH. The timing iscontrolled in part by the thickness of the first coating, which ispreferably sprayed onto the core. The variation in the amount of a firstcoating between tablets is preferably not more than 10% (between 90% and110%), based on the total weight of the first coating. More preferred,the variation in the amount of a first coating is not more than 5%(between 95% and 105%), based on the total weight of the first coating.Factors (process conditions) that may influence the intra-eninter-tablet uniformity of the first coating include, for example, panspeed, spray rate, spray pattern, nozzle type, viscosity, dryingtemperature, air flow rate and coating time, as is known to the skilledperson. When required, a temperature controlled curing step, for exampleheat treatment at 60-80° C. for 1-3 hours, is applied to the firstcoating after application, preferably spraying, of the first coating.

In addition, the amounts of the water-soluble and/or water-insolublehydrophilic substance in the first coating, and the identity of thewater-soluble and/or water-insoluble hydrophilic substance, furtherprovide means to modulate the timing of release of a first activeingredient. For example, a tablet comprising a pressed core and a firstcoating with an average thickness of about 35 micrometer, the coatingcomprising Ethocel 20 and lactose in a 3:2 ratio, provides release ofthe first active ingredient at about 36 minutes after hydration of thetablet, while the same composition of a tablet with a first coating withan average thickness of about 50 micrometer, provides release of thefirst active ingredient at about 84 minutes after hydration of thetablet. A tablet comprising a pressed core and a first coating with anaverage thickness of about 90 micrometer, the coating comprising Ethocel20 and Avicell PH102 in a 3:2 ratio, provides release of the firstactive ingredient at about 105 minutes after hydration of the tablet.The skilled person is able to generate a time controlled, immediaterelease drug delivery system according to the invention, based on theteaching and the examples provided in this application.

The total weight of a drug delivery device according to the invention ispreferably at least 50 milligram, more preferred at least 150 milligram,and preferably is between 50 and 500 milligram, more preferred between150 and 400 milligram, more preferred between 300 and 400 milligram,such as about 301.5 milligram, 325 milligram, or about 340 milligram.

A time controlled drug delivery system according to the inventionprovides release of a first active ingredient after about apredetermined period of time (lag time), such as after about 1 hourafter administration of the drug delivery system, more preferred afterabout 1.5 hours, more preferred after about 2 hours, more preferredafter about 2.5 hours, more preferred after about 3 hours, morepreferred after about 3.5 hours, more preferred after about 4 hours,more preferred after about 4.5 hours, more preferred after about 5hours, more preferred after about 6 hours, more preferred after about 7hours, more preferred after about 8 hours, more preferred after about 10hours, after administration of the drug delivery system.

The term “time controlled” drug delivery system refers to a drugdelivery system that provides release of a first active ingredient aftera predetermined period of time, for example 2 hours, whereby the releaseis independent of pH. The predetermined period of time is set and notdependent on the pH history in the gastro-intestinal tract.

The term “immediate release” drug delivery system refers to a drugdelivery system that provides release of a substantial amount of a firstactive ingredient within a predefined period of time. An immediaterelease drug delivery system, for example, provides the release of morethan 60% of a first active ingredient, more preferred more than 70%,more preferred more than 80%, within 30 minutes after rupture of thecoating, more preferred within 20 minutes, more preferred within 8minutes after rupture of the coating. Methods and means to determine theamount of a first active ingredient that is released from a drugdelivery system, and the time frame within which the ingredient isreleased, such as for example compendial dissolution methods, are knownto the skilled person such as, for example, United States Pharmacopoeia(USP) dissolution tests based on Apparatus 2 (the paddle method) andApparatus 3 (the reciprocating cylinder).

The immediate release of a first active ingredient is thought to becaused by moisture induced stress relaxation. The driving force for thisstress relaxation is the amount of stored energy within the core assurrounded by the polymer coating (Van der Voort Maarschalk et al.,1997. Int J Pharmaceutics 151: 27-34; Van der Voort Maarschalk et al.,1997. Pharm Res 14: 415-419; Steendam et al., 2001. J Control Rel 70:71-82; Laity and Cameron, 2010. Eur J Pharm Biopharm 75: 263-276).Stress relaxation mediates the breakage of a coated core according tothe invention in a nonlinear fashion. Hydration of the core and thehydrophilic substance in the first coating mediates stress relaxationsuch that an immediate burst of the coating after a predetermined periodof time is obtained. It was found that the presence of more than 6%(w/w) of a water-insoluble, gel-forming disintegrant interferes with theimmediate release of a first active ingredient and leads to moresustained release properties.

The term “first active ingredient” refers to the ingredient that ispresent in the core. Said first ingredient may be a single activeingredient or a mixture of two or more active ingredients. A firstactive ingredient that is present in the core of a drug delivery systemaccording to the invention can be any ingredient which is preferablyreleased after a defined period of time. Examples of active ingredientsthat are preferably released at a defined time after administration, forexample in the early morning, are anti-asthmatics (e.g.bronchodilators), anti-emetics, cardiotonics, vasodilators, anti-vertigoand anti-meniere drugs, anti-ulceratives, corticosteroids such asprednisone, other anti-inflammatory drugs, analgetics, anti-rheumatics,anti-arthritic drugs; anti-angina drugs; and anti-hypertensives. Inaddition, other compounds for which such formulations can be very usefulto improve patient compliance comprise sedatives such as diazepam,antidepressants, and other CNS compounds.

Other classes of active ingredients that are preferably formulated indrug delivery system according to the invention are bioactive proteins,peptides, enzymes, vaccines and oligonucleotides. Very often these typesof compounds are not resistant to the acidic environment of the stomach.

Yet a further preferred type of active ingredients that are preferablyformulated in a drug delivery system according to the invention is aningredient that is preferably administered in a biphasic release mode.The formulations of the present invention are particularly amenable toadministration of antibiotics such as penicillins, cephalosporins, andalso benzodiazepines, calcium antagonists and short-acting hypnotics.

Yet a further preferred type of active ingredients that are preferablyformulated in a drug delivery system according to the invention is adrug that is part of a medical combination of at least two differentactive ingredients. Embodiments of these types of active ingredients arecombinations of active ingredients, whereby a first active ingredient ismitigating the negative effects of a second active ingredient, orpromoting/enhancing the action of a second active ingredient. Examplesare second active ingredients that cause side effect such as, forexample, constipation, nausea, gas/bloating, heartburn, pain or cramps.A first active ingredient is provided in advance of the second activeingredient. The first active ingredient mitigates the above side effectof the second active ingredient, e.g. provides laxative medication,nausea treatment medication, anti-gas and anti-bloating medication,anti-acid medication, pain reliever & muscle relaxant medication.

Yet a further preferred example is provided by a first activeingredient, which is combined with a second active ingredient whichcontrols and stops the action of the first ingredient after the timenecessary for the action of the first ingredient. As an example, acombination of anti-cancer drug such as methotrexate with immediaterelease, and a “stopper” ingredient, such as L-leukovorin, with a timecontrolled release, can be advantageously delivered with a drug deliverysystem according to the invention. In all these examples, the secondactive ingredient is preferably formulated in a drug delivery systemaccording to the invention.

An even more preferred type of active ingredients that are preferablyformulated in a drug delivery system according to the invention isprovided by an active ingredient that acts synergistically with anotheractive ingredient in the same disease area, but which is to be releasedat a different time compared to the other active ingredient, and/or thathas to be administered at different areas in the oral and/orgastro-intestinal tract.

A most preferred example is a combination therapy preferably for thetreatment of male or female: sexual dysfunction, desire dysfunction, orerectile dysfunction. Preferably said combination treatment is treatmentof Hypoactive Sexual Desire Disorder. Preferably a combination oftestosterone or a functional analogue thereof and a first activeingredient is used, whereby the testosterone or a functional analoguethereof is provided such that the peak plasma level of testosteroneoccurs about 2-6 hours, more preferred 3-4 hours, prior to the peakplasma level of the first active ingredient. The first active ingredientis preferably provided in a time controlled, immediately release drugdelivery system according to the invention.

A preferred first active ingredient, preferably for treatment of thetreatment of male or female: sexual dysfunction, desire dysfunction, orerectile dysfunction, and preferably for the treatment of HypoactiveSexual Desire Disorder is selected from the group consisting of a PDE5inhibitor, an inhibitor of neutral endopeptidase (NEP) and a5-hydroxytryptamine 1A receptor agonist (5-HT1Ara). A PDE5 inhibitor ispreferably chosen from vardenafil, sildenafil and tadalafil or any ofthe other known PDE5-inhibitors. Further non-limiting examples of PDE5inhibitors are: E-4021, E-8010, E-4010, AWD-12-217 (zaprinast), AWD12-210, UK-343,664, UK-369003, UK-357903, BMS-341400, BMS-223131,FR226807, FR-229934, EMR-6203, Sch-51866, IC485, TA-1790 (avanafil),DA-8159 (udenafil), NCX-911 or KS-505a. Other examples can be found inWO 96/26940. A typical example for oral administration of vardenafil isprovided by vardenafil HCl which is designated chemically as piperazine,1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-/][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-4-ethyl-,monohydrochloride. Another example is given in sildenafil citrate whichis chemically designated as1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1Hpyrazolo[4,3-crlpyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazinecitrate.

A preferred PDE5-inhibitor according to the invention is sildenafilwhich is preferably administered as sildenafil citrate(1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1Hpyrazolo[4,3-crlpyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazinecitrate).

A further preferred first active ingredient for the treatment of male orfemale: sexual dysfunction, desire dysfunction, or erectile dysfunction,and preferably for treatment of Hypoactive Sexual Desire Disorder is aninhibitor of neutral endopeptidase (NEP).

A preferred NEP-inhibitor is selected from candoxatril; candoxatrilat;dexecadotril ((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycinebenzyl ester); CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155(N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680; JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-(3-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-(3-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-(3-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708; and combinations thereof.

A preferred NEP inhibitor according to the invention is selective forNEP (EC 3.4. 24.11) over soluble secreted endopeptidase (SEP). NEPdegrades a hormone called vasoactive intestinal peptide (VIP) thatpromotes blood flow to the vagina. Neuropeptides such as vasoactiveintestinal peptide (VIP) are major neurotransmitters in the control ofgenital blood flow. VIP and other neuropeptides are degraded/metabolisedby NEP. Thus, NEP inhibitors will potentiate the endogenous vasorelaxanteffect of VIP released during arousal. This will lead to enhancedgenital blood flow and hence genital engorgement. Selective inhibitorsof NEP enhance pelvic nerve-stimulated and VIP-induced increases invaginal and clitoral blood flow. In addition, selective NEP inhibitorsenhance VIP and nerve-mediated relaxations of isolated vagina wall.Therefore, the effects of a NEP-inhibitor are similar to the effects ofa PDE5-inhibitor, namely increased vaginal and clitoral blood flow.Preferred NEP inhibitors are UK-447,841 and UK-505,749.

A further preferred first active ingredient preferably for treatment ofmale or female: sexual dysfunction, desire dysfunction, or erectiledysfunction, and preferably for the treatment of Hypoactive SexualDesire Disorder is a 5-hydroxytryptamine 1A receptor agonist (5-HT1Ara).Preferably, a 5-HT1Ara is selective for the 5-HT1A receptor over other5-HT receptors and the α-adrenoreceptor and dopamine receptor.Non-limiting examples of a 5-HT1Ara are 8-OH-DPAT, Alnespirone, AP-521,Buspar, Buspirone, Dippropyl-5-CT, DU-125530, E6265, Ebalzotan,Eptapirone, Flesinoxan, Flibanserin, Gepirone, Ipsapirone, Lesopitron,LY293284, LY301317, MKC242, R(+)-UH-301, Repinotan, SR57746A,Sunepitron, SUN-N4057, Tandosporine, U-92016A, Urapidil, VML-670,Zalospirone and Zaprasidone. A preferred 5HT1A receptor agonist isbuspirone.

It is further preferred that a first active ingredient in a timecontrolled, immediate release drug delivery system according to theinvention is a combination of two or more active ingredients such as,but not limited to, two or more PDE5 inhibitors, two or more NEPinhibitors, two or more 5-HT1A receptor agonists, or a combination of atleast one PDE5 inhibitor and at least one NEP inhibitor, a combinationof at least one PDE5 inhibitor and at least one 5-HT1A receptor agonist,a combination of at least one NEP inhibitor and at least one 5-HT1Areceptor agonist, and a combination of at least one PDE5 inhibitor, atleast one NEP inhibitor and at least one 5-HT1A receptor agonist.

The invention further provides a dual drug delivery device, comprisingthe time controlled, immediate release drug delivery system according toinvention, wherein the first coating of the time controlled, immediaterelease drug delivery system is surrounded by a second coatingcomprising a second active ingredient.

The second coating provides release of the second active ingredient inan immediate release or a controlled release fashion. The second coatingmay be pressed or sprayed onto the outer surface of the first coating.Methods for pressing or spraying are known in the art. A second coatingthat surrounds the first coating advantageously protects the integrityof the first coating, for example during packaging or storage of a dualdrug delivery device. This will preferably decrease or minimize damageto the first coating occurring during packaging or storage that mighteffect the lag time of the release of the first active ingredient fromthe core of the dual drug delivery device.

The second coating is preferably sprayed onto the outer surface of thefirst coating. When a spray coat is used it is generally formulated tocontain a drug and film forming ingredient so that the drug is dispersedin the film that overlays the first coating of the core. Such filmforming ingredients are known in the art and may be for examplehydroxypropylmethylcellulose, povidone, hydroxyethylcellulose, othermodified celluloses known in the art, polyacrylates, polymethacrylates,and polymethyl/ethylmethacrylates. A film forming ingredient accordingto the invention preferably comprises hydroxypropylmethylcellulose, morepreferred low molecular weight hydroxypropylmethylcellulose with anumber average molecular weight below 20,000; more preferred below10,000.

The spray coat may be formulated to give a short sustained release byforming a coat that slowly dissolves or to give an immediate release byforming a coat that dissolves quickly. The amount of a film-formingingredient is preferably between 0.05 and 40% (w/w), based on the totalweight of the second coating, more preferred between 1 and 30% (w/w)such as, for example, about 20% (w/w).

The second coating preferably comprises a weight of between 0.5 and 5%(w/w) based on the total weight of the drug delivery device. Preferablysaid coating comprises a weight of between 1% and 3% and preferablybetween 1.5 and 2.5% (w/w) based on the total weight of the drugdelivery device. In a preferred embodiment the second coating of a drugdelivery system comprises a weight of between about 1-20 mg per unit.Preferably said second coating comprises a weight of about 3-15 mg perunit. In a particularly preferred embodiment said second coating of adrug delivery device of the invention comprises a weight of about 4-10mg per unit.

The second coating of a dual drug delivery device according to theinvention preferably comprises a second active ingredient. The amount ofa second coating that is sprayed onto the outer surface of the firstcoating therefore determines the amount of the second active ingredientin the dual drug delivery device. The amount of a second coating,therefore, needs to be controlled. The variation in the amount of asecond coating between tablets is preferably not more than 10% (between90% and 110%), based on the total weight of the second coating. Morepreferred, the variation in the amount of a second coating is not morethan 5% (between 95% and 105%), based on the total weight of the secondcoating. Factors (process conditions) that may influence the intra-eninter-tablet uniformity of the second coating include, for example, panspeed, spray rate, spray pattern, nozzle type, viscosity, dryingtemperature, air flow rate and coating time, as is known to the skilledperson. The amount of a second active ingredient is preferably between0.05 and 20% (w/w), based on the total weight of the second coating,more preferred between 0.5 and 10% (w/w).

Examples of known excipients that may be added to a sprayed or pressedsecond coating for controlled release are one or more polymers orcopolymers selected from acrylic and methacrylic acid polymers andcopolymers such as acrylic acid and methacrylic acid copolymers, methylmethacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, poly(acrylic acid), poly(methacrylic acid), methacrylicacid alkylamide copolymer, poly(methyl methacrylate), polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, poly(methacrylic acid anhydride), glycidylmethacrylate copolymers and ethylcellulose. The amount of knownexcipients is preferably below 10% (w/w), based on the total weight ofthe second coating, more preferred below 5% (w/w), more preferred below1% (w/w).

The second coating of a dual drug delivery device according to theinvention preferably provides immediate delivery of the second activeingredient in the mouth. The term “mouth” comprises the interspacebetween the lips and the teeth, the interspace between the cheek and theteeth, the oral cavity which is delimited by the palate and tongue andthe sublingual area. The second active ingredient is preferably releasedin the sublingual space in the mouth.

The term “immediate release of the second active ingredient” refers tothe rapid dissolution of the second coating in the mouth such that thesecond active ingredient is completely or substantially completelyreleased within a short time frame within the mouth. The term “immediaterelease of the second ingredient” indicates that at least 50% of thesecond active ingredient is released within 5 minutes, more preferredwithin 4 minutes, more preferred within 3 minutes, more preferred within2 minutes, most preferred within 1 minute after oral administration ofthe dual drug delivery device. It is more preferred that at least 70% ofthe second active ingredient is released within 5 minutes, morepreferred within 4 minutes, more preferred within 3 minutes, morepreferred within 2 minutes, most preferred within 1 minute after oraladministration of the dual drug delivery device.

An advantage of a dual drug delivery device according to the inventionis that food-effects are minimized. The term “food-effects” refers tothe difference in the rate and extent of absorption of a drug that isadministered shortly after a meal (fed conditions), as compared toadministration under fasting conditions. The release of the first activeingredient is not dependent on the pH and therefore not likely to beinfluenced by food effects. In addition, the formulation of the secondactive ingredient as an immediate release formulation also minimizesfood-effects for the release of the second active ingredient.

A further advantage of a dual drug delivery device according to theinvention is that it provides two independent dosing routes in onetablet.

A further advantage of a dual drug delivery device according to theinvention is that it provides first-pass free absorption into thesystemic circulation of one active ingredient (defined herein as secondactive ingredient) in combination with gastro-intestinal absorption of afurther active ingredient (defined herein as first active ingredient) inone tablet.

A further advantage of a dual drug delivery device according to theinvention is that it provides sublingual absorption into the systemiccirculation of one active ingredient (defined herein as second activeingredient) in combination with gastro-intestinal absorption of afurther active ingredient (defined herein as first active ingredient) inone tablet.

The second active ingredient may be similar or dissimilar to the firstactive ingredient. In one embodiment, a second active ingredient, forexample a steroid such as testosterone, is provided sublingually by adual drug delivery device according to the invention in the absence of afirst active ingredient. In this embodiment, the core of the dual drugdelivery device does not comprise an active ingredient.

The second active ingredient preferably is dissimilar to the firstactive ingredient. When the second active ingredient is dissimilar tothe first active ingredient, a further advantage of a dual drug deliverydevice according to the invention is that the timed release of the firstand second active ingredients avoids interactions that may occur betweenthe first and second active ingredient.

An example of a second active ingredient is methotrexate which isprovided in an immediate release formulation, and L-leukovorin which isprovided as a “stopper” ingredient in a time controlled, immediaterelease formulation.

Poorly soluble second active ingredients may be effectively absorbedfrom the mouth in the presence of a carrier. A suitable carrier forpoorly soluble active ingredients such as, for example, steroids such astestosterone, progesterone, and estradiol, NSAIDS, cardiac glycosides,antidiabetics or benzodiazepines comprises a cyclodextrin, a derivativethereof or a mixture of derivatives of cyclodextrin monomers or apolymer thereof. A derivative of a cyclodextrin is a chemicalmodification of a cyclodextrin at a hydroxyl site. A cyclodextrinpolymer is a chemical derivative where several cyclodextrin monomers orderivatives are covalently coupled. Oral administration of drugscomplexed with cyclodextrines or derivatives thereof led to effectiveabsorption and entry of the hormones into the systemic circulation,followed by gradual elimination, thus avoiding rapid first-pass loss.Suitable cyclodextrins are, for example,hydroxypropyl-beta-cyclodextrin, poly-beta-cyclodextrin andgamma-cyclodextrin, methyl-cyclodextrin and acetonyl hydroxypropylcyclodextrin.

A further example of a second active ingredient in a dual drug deliverydevice according to the invention is provided by estradiol or ananalogue or derivative thereof, for example for the treatment ofosteoporosis. Said estradiol or analogues thereof may be provided withone or more of an additional drug that is used in the treatment ofosteoporosis as a first active ingredient. An example of said additionaldrug is a calcium regulator such as alendronate, clodronate, etidronate,pamidronate, risedronate, tiludronate and/or ibandronate; a calcium saltsuch as, for example, calciumphosphate and/or calciumcarbonate; and/or avitamin D derivative such as, for example, cholecalciferol, calcitrioland/or alfacalcidol. Said estradiol or analogue or derivative thereofmay be replaced as a second active ingredient by a selective estrogenreceptor modulator (SERM), for example Raloxifene, or by parathyroidhormone, for example recombinant parathyroid hormone such asteriparatide. SERM and parathyroid hormone may also be provided with oneor more of an additional drug that is used in the treatment ofosteoporosis as a first active ingredient, as is indicated hereinabove.

A further example of a second active ingredient in a dual drug deliverydevice according to the invention is provided by nitroglycerin, forexample for the treament of angina pectoris. Oral, for examplesublingual, dosing of nitroglycerin is preferably combined with a timecontrolled, immediate release drug delivery system comprising one ormore of an additional angina drug as a first active ingredient. Saidadditional angina drug is preferably a beta-blocker such as, forexample, atenolol, pindolol, propranolol, oxprenolol, metoprolol and/orbisoprolol; a calcium antagonist such as, for example, amlodipine,diltiazem, nifedipine, bepridil, barnidipine, nicardipine and verapamil;and/or a selective heart-rate reducing ingredient such as, for example,ivabradine.

In a most preferred example, the second active ingredient istestosterone or a functional analogue thereof. This active ingredient ispreferably used in a therapy for treatment of male or female: sexualdysfunction, desire dysfunction, or erectile dysfunction, and preferablyfor the treatment of Hypoactive Sexual Desire Disorder. Preferably saidtherapy is a combination therapy together with a first activeingredient, whereby the testosterone or a functional analogue isprovided in an immediate release formulation in the second coating, anda first active ingredient is provided in the core of a time controlled,immediate release drug delivery system according to the invention.

The term “testosterone or functional analogue thereof” refers totestosterone or a precursor or metabolite of testosterone that providesthe same or a similar function as testosterone. Preferred precursors oftestosterone are selected from pregnenolone, 17α-hydroxypregnenolone,progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone,androstenedione, and androstenediol. Preferred metabolites oftestosterone are selected from hydroxyandrostenedione,hydroxytestosterone, including 2β-, 6β-, 7α-, 12α-, and16α-hydroxytestosterone, and dihydrotestosterone, including 5α- and5β-dihydrotestosterone. A preferred analogue of testosterone is capableof binding to an androgen receptor. It is most preferred that saidtestosterone or a functional analogue thereof is testosterone.

Said “testosterone or functional analogue thereof” in the second coatingis preferably combined with a PDE5-inhibitor, a NEP-inhibitor, and/or a5-HT1A receptor agonist. A dual drug delivery device, comprising a timecontrolled, immediate release drug delivery system comprising aPDE5-inhibitor, a NEP-inhibitor, and/or a 5-HT1A receptor agonistaccording the invention, wherein the first coating of the drug deliverysystem is surrounded by a second coating comprising testosterone orfunctional analogue thereof preferably provides the provision of thedrug delivery system comprising a PDE5-inhibitor, a NEP-inhibitor,and/or a 5-HT1A receptor agonist between 1.5-5 hours, more preferred 2-3hours, more preferred about 2.5 hours, after the provision oftestosterone or functional analogue thereof.

A second coating comprising a steroid such as testosterone or functionalanalogue thereof preferably comprises a carrier selected fromhydroxypropyl-beta-cyclodextrin, poly-beta-cyclodextrin,gamma-cyclodextrin and polyvinylpyrolidone. A preferredpolyvinylpyrolidone is low molecular weight polyvinylpyrolidone with amolecular weight of maximal 80000. A suitable polyvinylpyrolidone ispreferably selected from K10, K15, K25, K30, and K50. A most preferredcarrier is hydroxypropyl-beta-cyclodextrine. The presence of a poorlysoluble steroid such as testosterone and a carrier such as acyclodextrin provides rapid and efficient delivery of the testosteroneto the mucous membrane, from which the steroid is than rapidly absorbedinto the circulation. The amount of said carrier is preferably between0.5 and 70% (w/w), based on the total weight of the second coating, morepreferred between 2 and 60% (w/w), more preferred between 5 and 50%(w/w),

The second coating preferably comprises a flavouring compound inaddition to the second active ingredient and one or more excipients,such as, for example, a colouring agent. Said flavouring compound may beany natural, artificial or synthetic compound or mixture of compoundsthat is pharmaceutically acceptable. An illustrative list of flavoursfor pharmaceutical applications includes cyclic alcohols, volatile oils,synthetic flavour oils, flavouring aromatics, oils, liquids, oleoresinsand extracts derived from plants, leaves, flowers, fruits, stems, roots,and combinations thereof. Non-limiting examples of cyclic alcoholsinclude menthol, isomenthol, neomenthol and neoisomenthol. Non-limitingexamples of flavour oils include spearmint oil, cinnamon oil, oil ofwintergreen (methyl salicylate), peppermint oil, clove oil, bay oil,anise oil, eucalyptus oil, thyme oil, cedar leaf oil, oil of nutmeg,allspice, oil of sage, mace, oil of bitter almonds, cassia oil, andcombinations thereof. Suitable flavours also include, for example,artificial, natural and synthetic fruit flavours such as citrus oils(e.g., lemon, orange, lime, and grapefruit), fruit essences (e.g.,lemon, orange, lime, grapefruit, apple, pear, peach, grape, strawberry,raspberry, cherry, plum, pineapple, apricot or other fruit flavours).Other useful artificial, natural and synthetic flavours include sugars,polyols such as sugar alcohols, artificial sweeteners such as aspartame,stevia, sucralose, neotame, acesulfame potassium, and saccharin,chocolate, coffee, vanilla, honey powders, and combinations thereof.Other useful flavours include aldehydes and esters, such as benzaldehyde(cherry, almond), citral (lemon, lime), neral (lemon, lime), decanal(orange, lemon), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrusfruits), aldehyde C-12 (citrus fruits), tolyl aldehyde (cherry, almond),2,6-dimethyloctanal (green fruit), 2-dodenal (citrus mandarin), andcombinations thereof. A preferred flavouring compound is a cyclicalcohol such as, for example, menthol, isomenthol, neomenthol andneoisomenthol, preferably combined with an artificial sweetener such asaspartame. The amount of a flavouring compound is preferably between 0.1and 60% (w/w), based on the total weight of the second coating, morepreferred between 1 and 40% (w/w).

The presence of a flavouring compound in the second coating of a dualdrug delivery device according to the invention may mask a bitter orobjectional-tasting drug or excipient.

It is preferred that the flavouring compound in the second coating of adual drug delivery device according to the invention rapidly disappearsfrom the oral cavity. Sensing of the particular flavour in the oralcavity indicates to the user that the second coating has not completelydissolved and that the time controlled, immediate release drug deliverysystem which is encompassed within the second coating is to be held inthe mouth. During use, the second active ingredient is co-delivered withthe flavouring compound from the second coating. A subject can easilyrecognize that the device is delivering the second active ingredient dueto the presence of the flavour (taste). Eventually, the entire dose ofsecond active ingredient is delivered. At this point, the device alsostops delivering the flavour. The disappearance of the flavour (taste)indicates that the time controlled, immediate release drug deliverysystem may be swallowed.

The skilled person will understand that a flavouring compound may bepresent in the first coating, instead of in the second coating. In thatcase, the appearance of the flavour (taste) indicates that the timecontrolled, immediate release drug delivery system may be swallowed. Theskilled person will further understand that a first flavouring compoundmay be present in the second coating, while a second flavouring compoundis present in the first coating. Upon disappearance of the first flavour(taste), and tasting of the second flavour (taste), the subject knowsthat the device has delivered the entire dose of the second activeingredient.

It is further preferred that the roughness of the outer surface of thesecond coating differs from the roughness of the outer surface of thefirst coating in a device according to the invention. A subject can beinstructed to swallow the time controlled, immediate release drugdelivery system when a difference in roughness becomes evident. Thisprovides sufficient retention time of a device according to theinvention in the mouth so that the second active ingredient issufficiently released and absorbed.

The invention further provides the use of a flavouring compound in adual delivery drug device, for indicating that the device is to be heldin the mouth until the flavour (taste) has disappeared.

The invention further provides the use of a flavouring compound in adual delivery drug device, for indicating that the device is to be heldin the mouth until the flavour (taste) appears.

The invention further provides a method for preparing a dual deliverydrug device comprising a first and a second coating, whereby aflavouring compound is present in the second coating for indicating thatthe device is to be held in the mouth until the flavour (taste) hasdisappeared.

The invention further provides a method for preparing a dual deliverydrug device comprising a first and a second coating whereby a flavouringcompound is present in the first coating for indicating that the deviceis to be held in the mouth until the flavour (taste) appears.

The invention further provides the use of a difference in roughnessbetween an outer surface of a first coating and an outer surface of asecond coating in a dual drug delivery device for indicating that thedevice is to be held in the mouth.

The invention further provides the use of a difference in roughnessbetween an outer surface of a first coating and an outer surface of asecond coating in a dual drug delivery device for indicating that thedevice is to be swallowed.

The invention further provides a method for preparing a dual deliverydrug device comprising a first and a second coating, wherein a roughnessof an outer surface of the first coating differs from a roughness of anouter surface of the second coating.

In the present invention it was found that the active ingredient presentin the second coating of a drug delivery device as described hereinabove, is very well absorbed by the mucosa in the mouth. The absoluteabsorption as measured by bioavailability and the rate of absorptionwere significantly better when compared to a liquid with the same amountof active ingredient. Both variables where measured by measuring theconcentration of the active ingredient in the blood of the recipient atdifferent time points after administration. FIG. 11 depicts the resultsof a comparison of 0.5 mg testosterone in liquid form (F1) and with 0.5mg testosterone in a tablet of the invention (F2). The figure displaysthe concentration total testosterone (A) and free testosterone (B). Thecomposition of the tablet is given in table 7. The composition oftestosterone in liquid form is given in example 6. Both formulationswere held for a time period of 90 seconds under the tongue of healthyvolunteers. The depicted absorption profile was not expected. In theliquid phase the active ingredient is already dissolved whereas in thetablet the active ingredient is present as a solid that requiresdissolution prior to being available for absorption. This aspect isindependent from the presence of a first coating on the core. The firstcoating may be present or absent.

The invention therefore further provides a tablet for sublingualadministration of an active ingredient said tablet comprising a core,and a coating (outer coating) on the exterior surface of said core andoptionally a coating that separates said outer coating from said core(separation coating). In a preferred embodiment said outer coatingcomprises testosterone or a functional analogue thereof. In a preferredembodiment said core is a core as defined herein above for a timecontrolled immediate release drug delivery device. Preferably saidoptional separation coating is a first coating as identified hereinabove for a drug delivery device and preferably said outer coating is asecond coating as defined herein above for a dual drug delivery device.In a particularly preferred embodiment said outer coating comprises amixture of an active ingredient in amorphous form in an amount ofbetween about 0.1-10 mg; a coating polymer in an amount of between about0.25-25 mg; and water in an amount of between about 0.0-10% w/w of theouter coating. Said active ingredient in amorphous form is preferably asecond active ingredient as indicated herein above for a dual drugdelivery device. In a preferred embodiment said active ingredient inamorphous form is testosterone or a functional analogue thereof. Saidfunctional analogue of testosterone is preferably a functionaltestosterone analogue as defined herein above. In a particularlypreferred embodiment said active ingredient is testosterone. In thisembodiment said mixture preferably further comprises a cyclodextrin or apolyvinylpyrolidone or a combination thereof, in an amount of between0.25-25 mg. In a preferred embodiment said mixture comprises said activeingredient in an amount of between about 0.2-5.0 mg; said coatingpolymer in an amount of between about 0.5-12.5 mg; and water in anamount of between about 0.0-5% w/w of the outer coating. In thisembodiment said mixture preferably further comprises a cyclodextrin or apolyvinylpyrolidone or a combination thereof in an amount of between0.25-25 mg. Whereas the mixture may comprise cyclodextrin or apolyvinylpyrolidone or a combination thereof, it is preferred that saidmixture comprises cyclodextrin. Tablets with a mixture containingcyclodextrin and not polyvinylpyrolidone are more stable particularlywhen the active ingredient is testosterone or a functional analoguethereof. Both cyclodextrin and polyvinylpyrolidone prevent amorphoustestosterone or a functional analogue thereof from crystallizing in thesolid coating when exposed to prolonged incubation and/or varioustemperatures such as can occur during storage of the tablets. A coatingpolymer for said outer coating is preferably a film forming ingredientas indicated herein above for said second coating of a dual drugdelivery device. Said mixture preferably further comprises a sweetenerand/or a flavor as defined herein above. In a preferred embodiment saidouter coating consist of said mixture. A tablet of this embodiment may,as indicated herein above comprise a separation coating that separatessaid outer coating from said core. Said separation coating is, whenpresent, preferably a pH-independent coating or a pH-dependent coating,preferably an acid soluble coating or an enteric coating. In anotherpreferred embodiment said separation coating is a first coating asdefined herein above for a drug delivery device. Said separation coatingpreferably comprises a hydrophobic polymer and a hydrophilic substanceas defined herein above for a drug delivery device. In this preferredembodiment said core and said optional separation coating have a volumeof between 50-1000 mm³. Preferably said core comprises a cellulose asdefined herein above for a drug delivery device, a filler such as anorganic and/or inorganic salt as defined herein above for a drugdelivery device and an active ingredient. Preferably said activeingredient is a first active ingredient as defined herein above for adrug delivery device.

The invention further provides a method for administering an activeingredient to an individual said method comprising providing theindividual in need thereof with a dual drug delivery device or tabletaccording to the invention, wherein said individual holds the dual drugdelivery device or tablet in the mouth for between 10 seconds and 5minutes and wherein said individual subsequently swallows said dual drugdelivery device or tablet. In a preferred embodiment said individualholds the dual drug delivery device or tablet in the mouth for between30 seconds and 2.5 minutes prior to swallowing said dual drug deliverydevice or tablet. Preferably said individual holds the dual drugdelivery device or tablet in the mouth for 60 seconds to 90 secondsprior to swallowing said dual drug delivery device or tablet. In apreferred embodiment said dual drug delivery device or tablet is heldunder the tongue for the indicated time. In a particularly preferredembodiment, said dual drug delivery device or tablet is placed under thetongue, whereupon the individual gently holds or moves such as swishes,the dual drug delivery device or tablet about for 90 seconds. It ispreferred that said individual does not swallow the dual drug deliverydevice or tablet or saliva during the incubation period in the mouth andpreferably under the tongue. The individual preferably does not chew orbite on the dual drug delivery device or tablet. Upon completion of theincubation time the dual drug delivery device or tablet is preferablyswallowed as a whole by the individual, optionally together with a fluidsuch as water.

A dual drug delivery device or tablet comprising testosterone or afunctional analogue thereof in the outer coating or as a second activeingredient can favorably be used for the treatment of male or female:sexual dysfunction, desire dysfunction, or erectile dysfunction, andpreferably for the treatment of Hypoactive Sexual Desire Disorder. Theinvention thus further provides a dual drug delivery device or tablet ofthe invention, for sublingual administration of testosterone or afunctional analogue thereof for the treatment of male or female: sexualdysfunction, desire dysfunction, or erectile dysfunction, and preferablyfor the treatment of Hypoactive Sexual Desire Disorder, wherein saiddual drug delivery device or tablet comprises a core, and a coating(outer coating) on the exterior surface of said core and optionally acoating that separates said outer coating from said core (separationcoating), wherein said outer coating comprises said testosterone or afunctional analogue thereof.

In a further preferred embodiment, a dual drug delivery device or tabletcomprising testosterone or a functional analogue thereof in the outercoating or as a second active ingredient can favorably be used for thetreatment of male hypogonadism. The invention thus further provides adual drug delivery device or tablet of the invention, for sublingualadministration of testosterone or a functional analogue thereof for thetreatment of male hypogonadism, wherein said dual drug delivery deviceor tablet comprises a core, and a coating (outer coating) on theexterior surface of said core and optionally a coating that separatessaid outer coating from said core (separation coating), wherein saidouter coating comprises said testosterone or a functional analoguethereof.

In a further preferred embodiment, a dual drug delivery device or tabletcomprising estrogen and/or progesteron or a functional analogue thereofin the outer coating or as a second active ingredient can favorably beused for the treatment of female hypogonadism. The invention thusfurther provides a dual drug delivery device or tablet of the invention,for sublingual administration of estrogen and/or progesteron or afunctional analogue thereof for the treatment of female hypogonadism,wherein said dual drug delivery device or tablet comprises a core, and acoating (outer coating) on the exterior surface of said core andoptionally a coating that separates said outer coating from said core(separation coating), wherein said outer coating comprises said estrogenand/or progesteron or a functional analogue thereof.

A preferred dual drug delivery device according to the inventioncomprises: core:

-   -   between 100 mg and 150 mg, preferably between 109 mg and 126.5        mg, of Pharmacel pH102;    -   between 100 mg and 150 mg, preferably between 109 mg and 126.5        mg, of DicalciumPhosphate 0 aq;    -   between 25 mg and 100 mg, preferably between 35 mg and 70 mg, of        Sildenafil citrate;    -   between 10 mg and 20 mg, preferably about 12 mg of        Croscarmellose;    -   between 1 mg and 2 mg, preferably about 1.5 mg of        Magnesiumstearate;

First Coating

-   -   between 5 mg and 20 mg, preferably about 12.5 mg of Ethocel 20;    -   between 5 mg and 20 mg, preferably about 12.5 mg of Avicel pH        105;

Second Coating:

-   -   between 1 mg and 2 mg, preferably about 1.34 mg of HPMC 5 cps    -   between 2 mg and 3.5 mg, preferably about 2.66 mg of        HydroxyPropyl B-cyclodextrin;    -   between 0.1 mg and 1 mg, preferably between 0.25 mg and 0.5 mg        of Testosterone.

The second coating of said preferred dual drug delivery preferablyfurther comprises between 1 mg and 2 mg, preferably about 1.34 mg ofPeppermint-oil and between 0.5 mg and 1.5 mg, preferably about 1.0 mg ofAspartame.

A further preferred dual drug delivery device according to the inventioncomprises: core:

-   -   between 50 mg and 150 mg, preferably between 75 mg and 125 mg,        preferably about 97.5 mg of Pharmacel pH 200;    -   between 150 mg and 250 mg, preferably between 175 mg and 225 mg,        preferably about 201.5 mg of DicalciumPhosphate 0 aq;    -   between 1 mg and 20 mg, preferably between 5 mg and 15 mg,        preferably about 10 mg of Buspirone Hydrochloride;    -   between 10 mg and 20 mg, preferably about 13 mg of        Croscarmellose;    -   between 1 mg and 10 mg, preferably between 2 mg and 5 mg,        preferably about 4.4 mg of Magnesiumstearate;

First Coating

-   -   between 5 mg and 20 mg, preferably about 14.7 mg of Ethocel 20;    -   between 10 mg and 50 mg, preferably between 20 mg and 40 mg,        preferably about 29.3 mg of Avicel pH 105;

Second Coating:

-   -   between 1 mg and 2 mg, preferably about 1.34 mg of HPMC 5 cps    -   between 2 mg and 3.5 mg, preferably about 2.66 mg of        HydroxyPropyl B-cyclodextrin;    -   between 0.1 mg and 1 mg, preferably between 0.25 mg and 0.5 mg        of Testosterone.

The second coating of said preferred dual drug delivery preferablyfurther comprises between 1 mg and 2 mg, preferably about 1.34 mg ofPeppermint-oil and between 0.5 mg and 1.5 mg, preferably about 1.0 mg ofAspartame.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 In vitro release pattern of Ethocel coating. The figurerepresents the release profile of one tablet coated with a mixture ofEthocel 45 and lactose 200 mesh (11a). The burst at a lag time of 1.90h±12 min is equivalent to that of other coatings that are described intable 1-3. Within 6 minutes, more than 80% of the drug is released.

FIG. 2 Scanning electron microscopy (SEM) micrographs showing coatingsurface characteristics. The black dots are pores on the surface.

(A) Tablet coated with Ethocel/Avicel PH105 (1:1). There are multiplepores present before and

(B) after rupture.

(C) Ethocel/lactose 450 m (1:1) coating hardly contains any pores.

(D) multiple pores were formed when the coating was ruptured.

FIG. 3 SEM micrographs, showing a cross section of first coating beforerupture of the coating. (A) Ethocel/Avicel PH105 (1:1). (B)Ethocel/Lactose 450 m.

FIG. 4 Coat rupture time versus average coat weight of sildenafil coretablets as obtained in a perforated drum film coater. Data are for firstcoatings with 60% Avicel and 40% Ethocel (coat weight range 25-32 mgram)and for first coatings with 67% Avicel and 33% Ethocel (coat weightrange 34-46 mgram). Black lines: max values. Dark grey line: averagevalues. Light grey lines: min values.

FIG. 5 Testosterone assay versus weight of testosterone-comprisingsecond coat solution. The second coating solution was sprayed in aperforated drum film coater, indicating that the spray weight is asuitable endpoint for the coating process to obtain a proper contentuniformity for testosterone.

FIG. 6 Geometric mean total testosterone levels in serum afteradministration of 0.25, 0.50 and 0.75 mg sublingual testosterone.

Total testosterone normal range=0.14 to 0.66 ng/mL (0.5 to 2.3 nmol/L)(Davison et al., 2005). To convert total testosterone to nanomoles perliter, multiply by 3.467.

FIG. 7 Geometric mean free testosterone levels in serum afteradministration of 0.25, 0.50 and 0.75 mg sublingual testosterone.

Free testosterone normal range=0.00072 to 0.0036 ng/mL (2.5 to 12.5pmol/L) (Davison et al., 2005). To convert free testosterone topicomoles per liter, multiply by 3467.

FIG. 8 Free fraction of testosterone for 0.25 mg, 0.50 mg and 0.75 mgmeasured from t=4 min to t=30 min.

FIG. 9 Free fraction of testosterone for 0.25 mg, 0.50 mg and 0.75 mgmeasured from t=4 min to t=30 min for the low and high SHBG groups.

-   -   * significant difference between 0.25 mg vs. 0.75 mg (P=<0.05)    -   † significant difference between 0.25 mg vs. 0.50 mg (P=<0.05)

FIG. 10 Geometric mean DHT levels in serum after administration of 0.25,0.50 and 0.75 mg sublingual testosterone. DHT reference range=<0.29ng/mL (Davison et al., 2005). To convert total DHT to nanomoles perliter, multiply by 3.44.

FIG. 11 Comparison of testosterone bioavailability as measured by theuptake in blood of healthy individuals following administration oftestosterone in liquid formulation (F1) or the same amount (0.5 mg) oftestosterone in solid formulation (F2).

FIG. 12 Mean testosterone plasma concentration-time profiles measured inhealthy pre-menopausal female subjects.

FIG. 13 Mean free-testosterone plasma concentration-time profilesmeasured in healthy pre-menopausal female subjects.

FIG. 14 Mean sildenafil plasma concentration-time profiles measured inhealthy pre-menopausal female subjects.

FIG. 15 In vitro release pattern of individual sildenafil cores coatedwith 21.5 milligram of Ethocel/Avicel PH105 (1:1).

FIG. 16 In vitro release pattern of sildenafil from coated with 21.5milligram of Ethocel/Avicel PH105 (1:1).

EXAMPLES Materials and Methods Chemicals

Magnesium stearate; theophyline; crosscarmellose (AC-DI-SOL®); andethylcellulose (Ethocel 20, 45 (Standard premium)) were obtained fromDOW (Benelux). Microcrystalline cellulose (Avicel PH101, PH102, andPH105) and carboxymethylcellulosum-sodium (low viscous) were obtainedfrom OPG Farma. Maydis Amyllum was obtained from OPG Farma. Lactose 200mesh and 450 mesh (Pharmatose) was obtained from DMV-Fonterra.

Preparation of the Cores

Drug-containing core tablets were prepared by mixing 50 mg theophyline,12 mg Ac-Di-Sol, 119 mg microcrystalline cellulose (Avicel PH102) and119 mg Calcium phosphate. The core tablet excipients were blended for 15min in a Turbula-mixer, followed by the addition of magnesium stearate(0.5% w/w). The powder mixture was further mixed for 2 min. The coretablets (diameter, 9 mm; biconvex; hardness, 100 N; average tabletweight, 300 mg) were compressed at 10 kN.

Preparation of the Coating

Film coating was carried out in the bottom half of a florence flask witha rotational speed of 45 rpm. The flask was heated by hot air to ensureevaporation of the solvent. Prior to the coating process, the coretablets were heated for 45 minutes for dehydration. The solution ofethanol and Ethocel (3%), and the particulate material in suspension wascontinuously stirred to ensure a homogenous suspension. The suspensionwas sprayed onto the tablets at a speed of ˜1 ml/min. The weightincrease of the tablets was determined by weighing the tablets regularlyduring the coating process.

In Vitro Dissolution Tests

In order to establish how much drug is released from a formulation overtime, dissolution experiments were carried out using a USP dissolutionapparatus no. II (Prolabo, Rowa techniek BV) with a rotational speed of100 rpm and 500 ml of medium at 37° C. (n=5). The dissolution mediumthat was used comprised 0.1M phosphate buffer at pH 6.8. The amount oftheophylline dissolved was determined by UV absorbance at a wavelengthof 269 nm. The lag time was defined as the intersected point on the timeaxis when 25% of the drug in the tablets was released. FIG. 1exemplifies the burst pattern that was found for all coatings. After alag-time, more than 80% of the drug was released within 6 minutes,

Scanning Electron Microscopy

Scanning electron micrographs of the sections of the coating film ofpulsatile release tablets were taken before and after the dissolutiontest in pH 6.8 phosphate buffer using a scanning electron microscope(JEOL 6301F).

Example 1 Coating of Ethocel and Avicel

Theophyline containing cores were coated with Ethocel 20 (3%) anddifferent grades of Avicel (microcrystalline cellulose) in order toestablish a time controlled, immediate release of theophyline afterabout 2 hours. Avicel is widely used in many pharmaceuticalformulations. Avicel PH-105, PH-101 and PH-102 were examined since theyare chemically identical, yet they exhibit a range of particle sizes((nominal sizes are 20, 50 and 100 microns, respectively).

TABLE 1 In vitro lag times of tablets coated with Ethocel and Avicel.Coating composition Lag Ratio Weight Thickness ± S.D. Dissolved EthocelAgent (w/w) (mg/tablet) (μm) Average (min) (n = 5) 8  Ethocel Avicel 3:223.00 nd 1 h, 18 5 20 PH102 45 min 2  Ethocel Avicel 3:2 23.65 nd 1 h,14 5 20 PH101 54 min 3a Ethocel Avicel 3:2 16.01 60 2 h, 23 5 20 PH105 6 min 3b Ethocel Avicel 3:2 22.86 nd 3 h, >60  4 20 PH105 31 min 4aEthocel Avicel 1:1 21.12 nd 1 h, 13 5 20 PH105 41 min 4b Ethocel Avicel1:1 24.50 94 2 h, 15 5 20 PH105  2 min

The drug release lag times and corresponding coating formulations areprovided in Table 1.

The lag time is dependent on various variables. One of these variablesis the particle size. As shown in Table 1 Avicel 105 particles, with anominal size of 20 microns delay the rupture of the coating, compared toAvicel 102 and Avicel 101 particles (compare composition 3b withcompositions 2 and 8). This effect can be explained because particles of20 microns require increasing time for water to penetrate due toincreased hydrophobic interactions. This results in less capillaryaction and, hence, a decrease of the amount of water that is absorbed intime. This leads to a lower rate of water-transport into the inner coreand increases the lag time. A small particle size of themicrocrystalline cellulose also resulted in a greater variation of theresults.

The lag time is also dependent on the thickness of the coating asidentified by the weight of the tablet (compare composition 3b withcomposition 3a of Table 1). A thinner coating may allow the fluid topenetrate more easily into the core, resulting in a shortening of thelag time for disintegration. In addition, a thinner coating is lessrigid and disintegrates more easily, which also decreases the lag time.

A further parameter that affects the lag time is the ratio ofEthocel20/Avicel. A ratio of 1:1 instead of 3:2 (compare compositions(3b) and (4b) in Table 1) results in increased transport of water due toa larger amount of particles that transport water to the core. Thisreduces both the lag time and the observed variation of the results.Coating (2) with 100 micron Avicel particles and (4b) with 20 micronparticles have roughly the same weight and lag time but a differentratio of Ethocel/Avicel. Therefore, changing the ratio Ethocel/Avicelfrom 3:2 to 1:1 compensates the increase in lag time by the use ofsmaller Avicel particles. The advantage of using smaller particles isthat the coating suspension has better flow properties, which improvesthe overall film coating process.

The surface of the Ethocel/Avicel coating was inspected by scanningelectron microscopy (SEM). Multiple pores were found to be present bothbefore, and after rupturing (FIGS. 2 A and B). These pores channelthrough the coating, directly connecting the core to the outside, asshown in a cross-section of the coating (FIG. 3 A). It is likely thatthese pores are able to transport water directly into the core, next toor instead of transport via the Avicel particles.

Example 2 Coating of Ethocel and Lactose

A further framework for creating a pH-independent, time-controlledinflux of water into the core comprises a first coating withhydrophylic, water-soluble particulates within an hydrophobic layer.After a certain lag-time, the soluble component will be dissolvedleaving pores that can transport water into the core. This results indisintegration of the core, rupturing of the coating and release of thefirst active ingredient from the drug delivery system. The medium-influxis therefore also dependent on the dissolution-rate of lactose, inaddition to the diffusion-rate of medium trough the pores.

Lactose was chosen since there is a wide range of particle sizesavailable that can be useful as formulation variable. Lactose is adisaccharide that comprises galactose and glucose. Table 2 shows thedifferent formulations and the corresponding lag-times.

TABLE 2 In vitro lag times of tablets coated with Ethocel and lactose.Coating composition Lag Ratio Weight Thickness ± S.D. Dissolved ss #Ethocel Agent (w/w) (mg/tablet) (μm) Average (min) (n = 5)  8a Ethocel20 Lactose 450M 3:2  9.90  36 20 5  8b Ethocel 20 Lactose 450M 3:2 13.00 85 24 5  8c Ethocel 20 Lactose 450M 3:2 23.10 336 >60  2  9a Ethocel 20Lactose 450M 1:1 15.50  47  4 5  9b Ethocel 20 Lactose 450M 1:1 18.50 85 13 5  9c Ethocel 20 Lactose 450M 1:1 21.20  82 14 5  9d Ethocel 20Lactose 450M 1:1 26.20 115 >300  — 0 10a Ethocel 45 Lactose 450M 1:114.80  47  3 5 10b Ethocel 45 Lactose 450M 1:1 21.30 108 29 5 10cEthocel 45 Lactose 450M 1:1 24.50 143 >60  4 11a Ethocel 45 Lactose 200M1:1 17.90 114 12 5 11b Ethocel 45 Lactose 200M 1:1 21.6  >300  — 0

When the ratio of Ethocel/lactose 450 mesh is altered from 3:2 to 1:1,the overall number of pores that connect the outside of the coating tothe core will increase. Coatings with ratio of 1:1 (Ethocel/lactose), asopposed to 3:2, will allow the medium to diffuse faster to the innercore, which will cause the coating to rupture earlier and thus lower thelag time. This is shown in Table 2 with (8b) 13 mg coating; lag time of85 min (3:2) versus (9a), 15 mg coating; lag time 47 min (1:1). Anincreased amount of lactose in the coating resulted in less variationamong tablets (compare formulations (9) with formulations (8).

All Ethocel coatings containing lactose reach a weight-limit at whichthe coating won't rupture, for example 8c, 9c, 10c and 11b. The chanceof formation of pores that connect the outside of the coating with thecore becomes less when the coating is thicker. If the coating becomestoo tick, the chance of forming pores that connecting the outside of thetablet with the core is too small. Hence, no transport of water to thecore will occur, leaving the tablet intact.

A SEM micrograph of a tablet coated with Ethocel/lactose shows that theintact coating contains hardly any pores (FIG. 2 C), while the rupturedcoating reveals the formation of multiple pores (FIG. 2 D). Furthermore,a cross section of the coating (FIG. 3 B) shows that the intactEthocel/lactose-coating contains hardly any pores, unlike theEthocel/Avicel coating (FIGS. 3 B and A respectively)

Example 3 Preparation of Preferred Drug Delivery Systems Preparation ofthe Core Materials

-   -   Crosscarmellose, ViVaSol, JRSPharma, Ph. Eur., batch 9907    -   DiCalciumPhosphate anhydrous, Budenheim, USP.    -   MagnesiumStearate, Bufa, Ph. Eur, lot 04j22fs    -   Pharmacel PH102, DMV-Fonterra, Veghel    -   Sildenafil citrate

All materials, except for magnesium stearate, were mixed for 15 minutesusing a Turbula mixer at 90 rpm. After adding the magnesium stearate,the mixture was further mixed for 2 minutes.

Tablets were prepared using an instrumented excenter press (HOKO), witha 9 mm biconcave die set. The compaction force was 10 kN. The tabletweight was about 300 mg.

TABLE 3 Compositions of the core: Sildenafil 50 mg Sildenafil 25 mgPharmacel PH102 109 mg 126.5 mg   DicalciumPhosphate 0 aq 109 mg 126.5mg   Sildenafil citrate  70 mg 35 mg Croscarmellose  12 mg 12 mgMagnesiumstearate 1.5 mg 1.5 mg  Total 301.5 mg  301.5 mg   Crushingstrength ~100N ~100N Disintegration time ~10 s ~10 s

Coating of the Core Materials

-   -   Ethocel 20, Dow Benelux, lot KI 19013T02    -   Avicel PH 105, FMC, Ph. Eur, lot. 50750C

Preparation of First Coating Solution

-   -   A solution of 50 ml containing 3% Ethyl cellulose (=1.5 g Ethyl        cellulose) was prepared in ethanol 96%. 1.5 g Avicel PH 105 was        added to the suspension.

The first coating solution was sprayed with a nozzle (0.7 mm internaldiameter) on a batch of tablets inside a small spraying-vessel (glass).The suspension was stirred during the whole process. During theprocedure, the spraying-vessel was heated with hot air to evaporate thesolvent. The coating process was stopped when about 25 mg Ethylcellulose/Avicel per tablet was sprayed.

Example 4 Preparation of Preferred Dual Drug Delivery Devices Materials

-   -   Testosterone, Sigma    -   HPMC 5 cps Ph. Eur Sigma-Aldrich, lot. 12816TD    -   Hydroxypropyl-beta cyclodextrin M.S.=0.8, Aldrich, Ph. Eur, lot        30638-089    -   Peppermint oil, Bufa, Ph. Eur, lot.09j16-B01    -   Aspartame, Bufa, Ph. Eur, lot.02a17fr

Preparation of Solutions

5% HPMC-solution: 5 g HPMC 5 cps was dissolved in 85 ml Ethanol 96%+15ml demi-water

5% HPBCD-solution: 5 g HPBCD was dissolved in 100 ml Ethanol 96%.

1% Peppermint-oil: 1 g Peppermint-oil was dissolved in 100 ml Ethanol96%

Second Coating Solution

6.7 ml 5% HPMC solution = 0.335 g HPMC 5 cps 13.3 ml 5% HPBcd solution =0.665 g HydroxyPropyl B-cyclodextrin 30 ml 1% peppermint-oil solution = 0.3 g Peppermint-oil 0.250 g Aspartame = 0.250 g Aspartame 0.125 gtestosterone = 0.125 g Testosterone 20 ml demi-water Total volume: 70 ml

The second coating solution was sprayed with a nozzle (0.7 mm internaldiameter) on a batch of tablets comprising a core and first coating asshown in example 3. Spraying was performed inside a smallspraying-vessel (glass). The vessel was heated with hot air to evaporatethe ethanol. The coating process was stopped until 0.5 mgtestosterone/tablet (6.7 mg total weight) was sprayed.

TABLE 4 Composition of second coating of dual drug delivery devicesSildenafil 50/25 mg Sildenafil 50/25 mg Testosterone 0.5 mg Testosterone0.25 mg HPMC 5cps 1.34 mg 1.34 mg HydroxyPropyl B- 2.66 mg 2.66 mgcyclodextrin Peppermint- oil  1.2 mg  1.2 mg Aspartame  1.0 mg  1.0 mgTestosterone 0.50 mg 0.25 mg Total final coating 6.70 mg 6.45 mg

TABLE 5 Preferred dual drug delivery devices Sildenafil 50 mg Sildenafil25 mg Sildenafil 50 mg Sildenafil 25 mg Testosterone TestosteroneTestosterone Testosterone  0.5 mg  0.5 mg  0.25 mg  0.25 mg Pharmacel pH102   109 mg 126.5 mg   109 mg 126.5 mg DicalciumPhosphate 0 aq   109 mg126.5 mg   109 mg 126.5 mg Sildenafil citrate   70 mg   35 mg   70 mg  35 mg Croscarmellose   12 mg   12 mg   12 mg   12 mgMagnesiumstearate.  1.5 mg  1.5 mg  1.5 mg  1.5 mg Total core 301.5 mg301.5 mg 301.5 mg 301.5 mg Ethocel 20  12.5 mg  12.5 mg  12.5 mg  12.5mg Avicel pH 105  12.5 mg  12.5 mg  12.5 mg  12.5 mg HPMC 5cps  1.34 mg 1.34 mg  1.34 mg  1.34 mg HydroxyPropyl B-cyclodextrin  2.66 mg  2.66mg  2.66 mg  2.66 mg Peppermint-oil  1.2 mg  1.2 mg  1.2 mg  1.2 mgAspartame  1.0 mg  1.0 mg  1.0 mg  1.0 mg Testosterone  0.50 mg  0.50 mg 0.25 mg  0.25 mg Total second coating  6.70 mg  6.70 mg  6.45 mg  6.45mg Grand total 333.2 mg 333.2 mg   333 mg   333 mg

TABLE 6 Preferred dual drug delivery devices Sildenafil 50 mg Sildenafil25 mg Sildenafil 50 mg Sildenafil 25 mg Testosterone TestosteroneTestosterone Testosterone 0.5 mg 0.5 mg 0.25 mg 0.25 mg Pharmacel pH 200101.5 mg   119 mg 101.5 mg   119 mg DicalciumPhosphate 0 aq 101.5 mg  119 mg 101.5 mg   119 mg Sildenafil citrate   70 mg   35 mg   70 mg  35 mg Croscarmellose   12 mg   12 mg   12 mg   12 mgMagnesiumstearate.   15 mg   15 mg   15 mg   15 mg Total core   300 mg  300 mg   300 mg   300 mg Ethocel 20  12.5 mg  12.5 mg  12.5 mg  12.5mg Avicel pH 105  12.5 mg  12.5 mg  12.5 mg  12.5 mg HPMC 5cps  1.34 mg 1.34 mg  1.34 mg  1.34 mg HydroxyPropyl B-cyclodextrin  2.66 mg  2.66mg  2.66 mg  2.66 mg Peppermint-oil  1.2 mg  1.2 mg  1.2 mg  1.2 mgAspartame  1.0 mg  1.0 mg  1.0 mg  1.0 mg Testosterone  0.50 mg  0.50 mg 0.25 mg  0.25 mg Total second coating  6.70 mg  6.70 mg  6.45 mg  6.45mg Grand total 331.7 mg 331.7 mg 331.7 mg 331.7 mg

Example 5 Preparation of Preferred Dual Drug Delivery Device

Sildenafil citrate, dicalcium phosphate anhydrous, microcrystallinecellulose and croscarmellose were combined in a container and mixed. Themixture was passed through a 600 micron mesh into a blending container.The blend was tumbled for 30 minutes. Magnesium stearate was passedthrough a 600 micron mesh and added to the blend. The blend waslubricated by tumbling for up to 10 minutes. The blend was then placedin a tablet machine equipped with 9 mm biconcave punches and compressedto a tablet weight of 300 mg.

Ethylcellulose and microcrystalline cellulose were dispersed in ethanoland uncoated tablet cores were loaded into a perforated drum filmcoater. The dispersed ethylcellulose and microcrystalline cellulose weresprayed onto the cores and the solvent was removed by heat. The tabletswere cooled gradually in the coater prior to the next coating step.

Hydroxypropyl beta-cyclodextrin was dispersed in water. Testosterone wasdissolved in ethanol. After addition of the organic and aqueous phase,stirring was performed to allow the testosterone to interact with thecyclodextrin. Aspartame, menthol and hydroxypropyl methylcellulose(hypromellose) were added and stirring was continued. The resultantsuspension was sprayed onto the coated core tablets described above in aperforated drum coating pan. The solvent was removed by heating withair.

According to this procedure, tablets were made with various coat rupturetimes by modification of the first coating composition and first coatweight as shown in FIG. 4. For this, cores were coated either withweights of 25.7, 29.0 and 31.2 mg of 60% Avicel and 40% Ethylcellulose,or with weights of 34.3, 40.9 and 45.3 mg of 67% Avicel and 33%Ethylcellulose.

FIG. 5 indicates that for determining the end point for the coatingprocess with the testosterone coat the weight of the second coatingsolution sprayed is an excellent indicator for the total amount oftestosterone applied to the tablets. The testosterone content uniformityof three batches as described in FIG. 5 was well within Pharmacopeialrequirements with relative standard deviations of 4.2, 2.8 and 3.1% forbatches MOR202/66, /71 and /75 respectively.

Example 6

Context: Sublingual testosterone is a single-dose treatment often usedin studies regarding social, cognitive and sexual behavior. It ishypothesized that an increase in the ratio of free to total testosterone(free fraction) is indirectly, via genomic effects, responsible for thebehavioral effects after sublingual testosterone administration.

Objective: To characterize the pharmacokinetics of three dosessublingual testosterone in premenopausal women. Also, to investigate theSHBG saturation threshold influencing the free level and free fractionof testosterone.

Design: We conducted an investigator-blind, randomized, cross-overplacebo controlled study.

Setting: This study was undertaken at the research and developmentdepartment of a scientific company for research regarding female sexualdysfunction.

Participants: 16 healthy premenopausal women (mean age 27.3±5.3 yr).

Interventions: Sublingual testosterone solution; 0.25, 0.50 and 0.75 mg.

Main Outcomes Measure: The pharmacokinetics of three single dosessublingual testosterone solution; the influence of SHBG levels on freeand total levels of testosterone.

Results: After sublingual testosterone administration, serum free andtotal testosterone levels peaked at 15 min. and reached baseline levelswithin 150 min. The AUCs and Cmax of free and total testosteronediffered significantly between the three doses (P<0.0001) and increaseddose-dependently.

A dose-dependent increase in free fraction of testosterone was found inwomen with low SHBG levels, but not in women with high SHBG levels.

Conclusions: The three doses sublingual testosterone are rapidlyabsorbed and quickly metabolized in premenopausal women. These datademonstrate the influence of SHBG levels on the treatment inducedalterations in plasma free testosterone.

Introduction

Results of scientific research indicate that testosterone is involved insocial behavior (Bos et al., 2010; Eisenegger et al., 2010), includingsexual behavior (Auger, 2004; Hull and Dominguez, 2007). Sexual behavioris influenced by endogenous testosterone levels as well as toexogenously administered testosterone. For exogenous testosteroneadministration, two different methods of treatment can be distinguished:chronic treatment versus single dose administration. Each method oftreatment has its own pharmacokinetic profile, which may affect theinfluence of testosterone on behavior. Chronic testosteroneadministration is utilized as the delivery option in the majority ofstudies regarding the influence of testosterone on women's sexualbehavior, including hormonal replacement therapy in naturally orsurgically (bilateral oophorectomy) menopausal women (Sherwin, 2002;Shifren et al., 2000; Simon et al., 2005).

More recently however, several studies have investigated the effects ofsingle dose testosterone administration on women's sexual behavior(Tuiten et al., 2000; Tuiten et al., 2002; van der Made et al., 2009).Tuiten et al. reported that a single sublingual dose of 0.50 mgtestosterone significantly increased vaginal vasocongestion andexperiences of sexual lust and genital sensation in premenopausal womenwithout sexual complaints (Tuiten et al., 2000). These effects occurred3 to 4½ h after the induced testosterone peak and about 2½ h aftertestosterone returned to baseline levels. This delay in behavioraleffects after sublingual testosterone administration has been replicatedin several other studies regarding social behavior and cognitivefunctions (Aleman et al., 2004; Bos et al., 2010; Eisenegger et al.,2010; Hermans et al., 2006; Hermans et al., 2007; Hermans et al., 2008;Postma et al., 2000; Schutter and van Honk, 2004; van Honk et al., 2001;van Honk et al., 2004; van Honk et al., 2005; van Honk and Schutter,2007).

There are very few studies that have defined the pharmacokinetic profileof sublingual testosterone. Salehian et al. (Salehian et al., 1995),compared the pharmacokinetic profiles of 2 doses of sublingualtestosterone (2.5 and 5.0 mg) with the pharmacokinetic profile of along-acting testosterone ester, testosterone enanthate (TE) (in oil, im.200 mg) in hypogonadal men. Compared to sublingual testosterone, thetotal and the free testosterone levels peaked days later in the malesubjects studied who received TE. In the sublingual conditions the riseof free testosterone levels occurred within 1 h after administration, inthe TE group this occurred 7 days after administration. Furthermore, itwas shown that the free testosterone levels in the TE condition did notincrease until the sex hormone binding globulin (SHBG) levels weresuppressed after administration by day 7. The suppression of SHBG levelswas significantly greater in the TE group than in either sublingualgroup (Salehian et al., 1995).

It is widely accepted that free testosterone is the biologically activetestosterone (Mendel, 1989). Pharmacodynamic effects (measures of sexualfunctioning) would thus be expected to increase much later in the TEadministered group compared to the sublingual administered group.Unfortunately, in the Salehian et al. study, post-dose sexual motivationwas measured for the first time in the week before the first visit onday 20, when the free testosterone rise had already been passed in bothgroups. Notably, in the study by Tuiten and Van der Made et al.,measures of sexual arousal increased 3½-4 h after the peak ofcirculating testosterone (Tuiten et al., 2000; van der Made et al.,2009) and 2.5 hours after testosterone levels returned to baseline(Tuiten et al., 2000), indicating that sublingual testosteroneadministration produces a pharmacodynamic effect after 4 h. Van der Madeet al. suggested a SHBG saturation threshold hypothesis; i.e., whenavailable binding sites of SHBG are occupied with testosterone after asufficient single sublingual dose of testosterone, free fraction-, andthus free testosterone levels increase thereby inducing behavioraleffects (van der Made et al., 2009). The exact mechanism responsible forthis delay in behavioral effect is not fully understood but it could bethat testosterone exerts its behavioral effect via androgenicmetabolites, genomic mechanisms (Bos et al., 2011) or a combination ofthese factors.

The main purpose of the present study was to establish an extensivepharmacokinetic profile of three different single doses of sublingualtestosterone administered as a solution with cyclodextrin. The primarypharmacokinetic endpoints were levels of total and free testosterone.Secondary endpoints included the pharmacokinetics of5α-dihydrotestosterone (DHT), and 3α-androstanediol glucuronide(3α-diol-G). Additionally serum albumin, 17β-estradiol (E₂) and SHBGwere measured.

Moreover, we compared the data of the present study with those of theTuiten et al. pharmacokinetic study (Tuiten et al., 2000) with regard tothe effect of single dose sublingual testosterone on circulating freeand total testosterone levels. Furthermore we sought to determine atwhich level serum testosterone occupies the available binding sites ofSHBG and serum free testosterone increases, i.e., the postulated SHBGsaturation threshold mechanism by van der Made et al. (van der Made etal., 2009).

Subjects and Methods Study Subjects

Eligible women were between 21 and 40 years, premenopausal and had abody mass index (BMI) between 18 and 30 kg/m². Exclusion criteriaincluded a history of a hormone-dependent malignancy, endocrine disease,neurological problems, psychiatric disorder, cardiovascular condition,hypertension, abnormal liver or renal function. Women taking medicationsthat interfere with metabolism of sex steroids or had used testosteronetherapy within 6 months before study entry were excluded also.

Women were recruited and enrolled from referrals, newspaperadvertisements, the internet, and an internal database of our lab. Todetermine eligibility, participants were screened two weeks prior tostudy entry. In addition to an assessment of medical history, allsubjects received a physical examination including a 12-leadelectrocardiogram, standard biochemistry and hematological laboratorytests. Blood samples for determination of testosterone, SHBG, TSH,Thyroxine, FSH and estrogen were collected at baseline. A urinepregnancy test was applied to all women of child bearing potential.

16 healthy young women participated after providing written informedconsent and received reimbursement for expenses for their participation.This study was approved by the local ethics committee (StichtingTherapeutische Evaluatie Geneesmiddelen Medisch EthischeToetsingscommissie, Almere, The Netherlands) and carried out inagreement with ICH-GCP (International Conference on Harmonization—GoodClinical Practice).

Study Design

This was a single-center, investigator-blind, randomized, cross-overplacebo controlled study with three doses of a testosterone solutioncontaining cyclodextrin administered sublingually. This solutionconsists of authentic nonmodified testosterone forming a soluble complexby a cyclodextrin carbohydrate ring. Due to increased solubility theabsorption of testosterone through the oral mucosa is facilitated,thereby avoiding the hepatic first-pass metabolism (Brewster et al.,1988; Salehian et al., 1995; Stuenkel et al., 1991; Zhang et al., 2002).

All 16 subjects received each investigational drug dose once in randomorder. Wash-out between treatments was at least 48 h. Subjects hadserial blood samples drawn via an intravenous catheter. Pharmacokineticparameters were monitored at baseline and (at 2, 4, 6, 8, 10, 20, 30,60, 90, 120, 180, 230 min) after dosing.

Measurement of total testosterone, free testosterone, and DHT wereperformed at each sampling time; E₂ at −5, 60 and 230 min; 3α-diol-G at−5, 60, 120, and 230 min; SHBG and albumin prior to dosing and at 230min. Blood samples in the placebo condition were only measured at −5,10, 60 and 230 min.

Vital signs were measured at regular intervals and an electrocardiogramwas performed prior to dosing and at the end of the experimental day.For each experimental day, subjects were asked to attend the visit infasting state and they received a strict diet (low fat, no caffeine)during the experimental day to minimize the influence of pharmacokineticparameters. Drug, alcohol and pregnancy screens were performed prior toexperimental sessions.

Medication and Dosing

Testosterone and placebo were administered sublingually in 4 separateexperimental phases with either a 0.25, 0.50, 0.75 mg dose and placeboas a solution using a micropipette (Gilson Pipetman P1000) from a 1mg/ml solution. The 0.25 mg, 0.50 mg, and 0.75 mg testosterone weredosed from different volumes of the 1 mg/ml solution. For the placebosolution 0.50 ml was administered.

The different doses were prepared by an unblinded research associate andadministered by blinded research associates. The blinded researchassociate administered the solution into the subjects mouth under thetongue, the subjects were instructed to keep the solution sublinguallyfor 1 minute while moving the tongue slightly to optimize absorption.After 1 minute the blinded research associate instructed the subject toswallow the solution.

Hormone Assays

The assay used for the determination of total testosterone, freetestosterone (after ultrafiltration), and DHT was High PerformanceLiquid Chromatography with Mass Spectrometric detection (LC/MSMS) (API4000, AB Sciex). The method was validated with a lower limit ofquantification (LLOQ) of 0.02 ng/mL for testosterone and DHT, and 0.001ng/mL for free testosterone. The LC/MSMS assay is a reliable method foranalysis of free testosterone and overcomes the known limitations ofdirect immunoassays in measurement of testosterone values in the lowerrange (Labrie et al., 2006; Miller et al., 2004).

E₂ was analysed by a chemiluminescence immunoassay (Siemens), the LLOQwas 0.25 pmol/L. 3α-diol-G was measured by ELISA (BioVendor), the LLOQwas 0.25 ng/mL. SHBG was measured by an electrochemiluminescent assay(ECLIA, Roche). Albumin was measured by Roche Bromocresol Green (BCG)analysis (Roche).

Statistical Analysis

The pharmacokinetic parameters were analyzed using the WinNonlinsoftware (version 5.1). Pharmacokinetic parameters including area underthe curve, t=0 till t=230 min (AUCO-230), maximum concentration (Cmax)and time to maximum concentration (tmax) were calculated based on actualand baseline corrected individual concentration time curves. AUCs wereestimated using the linear trapezoidal rule. Individual pharmacokineticparameters AUCO-230 and Cmax and corresponding dose normalizedparameters were log transformed and analyzed using a mixed maximumlikelihood analysis (PROC MIXED in SAS, version 9.1) including subjectas a random factor and drug as a fixed effect factor. Contrasts weremade of the least square means to compare the different doses. Tmax wasanalyzed using a Wilcoxon rank sum test. This was based on the plannedtimes corresponding to the actual tmax to prevent bias in analysisresults based on differences in sampling times.

The baseline levels of total and free testosterone, DHT, E₂, 3α-diol-G,SHBG and albumin were calculated by taking the mean of the placebo,0.25, 0.50 and 0.75 mg predose levels.

Overall analysis of the free fraction (free testosterone levels dividedby total testosterone levels at each time point) was analyzed in a 3Drug (0.25 mg vs 0.50 mg vs 0.75 mg)×6 Time (t=4, 6, 8, 10, 20, 30 min.)repeated measures ANOVA, with Drug and Time as within subjects factors.

In order to meet normality assumptions, baseline SHBG values werelog-transformed and Pearson's correlation coefficients were calculatedto further investigate relationships between SHBG levels, totaltestosterone, free testosterone and free fraction percentage oftestosterone.

Subsequently, we divided the subjects into two subgroups, on the basisof their baseline SHBG levels (mean of placebo, 0.25, 0.50, 0.75 mgpredose levels). This subdivision was based on a median split of thebaseline SHBG levels. One group (N=8) with low SHBG levels (<63 nmol/L)and the other group (N=8) with relatively high SHBG levels (>63 nmol/L).Independent samples t-test were used to assess free testosterone levelswith SHBG as grouping variable (low vs. high SHBG) for each post-dosemeasurement.

The dependent variable free fraction was analyzed in a 3 Drug (0.25 mgvs. 0.50 mg vs. 0.75 mg)×6 Time (t=4, 6, 8, 10, 20, 30 min)×2 Group(SHBG low vs. SHBG high) repeated measures ANOVA, with Drug and Time aswithin subjects factor and Group as between subjects factor. To analyzethe effects of the within subject factors within each group separately,paired-samples t-test were used for each SHBG group for each post-dosemeasurement between the three doses. For all ANOVAs sphericity was notviolated. For all analyses a (two-sided) p-value less than 0.05 wasconsidered statistically significant. SPSS 16.0 was used for allstatistical analyses.

Results

The baseline characteristics and hormone levels of the 16 studyparticipants are outlined in table 8. One subject was excluded from the0.50 mg analysis due to an incorrect administration procedure of thetestosterone solution.

Primary Pharmacokinetic Endpoints

The pharmacokinetic parameters of total and free testosterone aresummarized in tables 9 and 10.

Total Testosterone

The three doses (0.25, 0.50, 0.75 mg) produced maximum levels of totaltestosterone of 3.79, 5.31 and 6.73 ng/mL, respectively, at means of15.6, 15.1 and 14.3 min (FIG. 6).

The Cmax of total testosterone was significantly different (P<0.0001)among the three doses. We found no statistically significant differencesin Tmax of total testosterone between the three dosages. The AUCs oftotal testosterone were also statistically significant different amongthe three doses (P<0.0001) and showed a dose-dependent increase. Thecalculated half-life of total testosterone showed a significantdifference between the 0.50 mg and 0.75 mg dose (P=0.125).

Free Testosterone

Peak levels for free testosterone during the three dosages were 0.021,0.032 and 0.043 ng/mL at means of 15.6, 14.4 and 12.8 min respectively(FIG. 7). There was a statistically significant difference between thethree doses with respect to Cmax of free testosterone (P<0.0001). Therewere no statistically significant differences for free testosterone Tmaxbetween the three dosages. Free testosterone AUCs were statisticallysignificant different between the three doses and increaseddose-dependently. The differences between the free testosterone AUCs ofthe 0.25 mg vs 0.50 mg and 0.25 mg vs 0.75 mg have P values <0.0001,while the difference between the 0.50 and 0.75 mg was significant atP<0.01. There were no statistically significant differences between thethree doses for the calculated half-life of free testosterone.

For all doses, baseline levels for total- and free testosterone werereached by 150 min.

Bioavailability

To determine the absolute percentage of the sublingual testosterone dosewhich is absorbed in the systemic circulation, the fraction of absorbedtestosterone needs to be calculated from the formula used also for theAUC calculation after intravenous dosing. Since we did not have anintravenous standard, we took the 0.25 mg dosage as reference value.Thus the bioavailability of the 0.25 mg was set at 100%, and for 0.50and 0.75 mg were calculated as 69% (or 0.34 mg), and 58% (or 0.43 mg),respectively. The bioavailability of sublingual testosteroneadministration decreases with increasing doses.

Free Fraction

Our analyses showed a statistically significant effect of drug dose onthe free fraction of testosterone (i.e. the ratio of free to totaltestosterone) during the t=4 through t=30 min measurements (P=0.002). Wealso found a statistically significant difference for the Cmax duringt=4 through t=30 min between the 0.25 mg and 0.50 mg (P=0.003) andbetween 0.25 mg and 0.75 mg doses (P=0.010), but not between the 0.50and 0.75 mg dose (P=0.381) (FIG. 8).

As stated above, we expected to find a relationship between circulatingSHBG and the increases in the free levels and the free fraction oftestosterone induced by the different dosages of sublingualtestosterone. Moreover, our experimental manipulations produced nostatistically significant changes in SHBG and albumin levels between andon test days (data not shown).

In our study population we found a large between-subject variation incirculating SHBG levels. Baseline SHBG levels (log transformed) werecorrelated with total testosterone levels (t=20 min): r=0.732, p<0.0002;r=0.930, p<0.001 and r=0.894, p<0.001 for the 0.25 mg, 0.50 mg and 0.75mg dose respectively. Baseline SHBG levels (log transformed) wereinversely correlated with free testosterone levels (t=20 min): r=−0.702,p<0.003; r=−0.849, p<0.001 and r=−0.798, p<0.001 for the 0.25 mg, 0.50mg and 0.75 mg dose respectively. For the free fraction levels and SHBGlevels, we observed stronger correlations; r=−0.947, p<0.001; r=−0.938,p<0.001 and r=−0.944, p<0.001 for the 0.25 mg, 0.50 mg and 0.75 mg doserespectively on t=20.

Because of this large between-subject variation we subdivided thesubjects in two group based on a median split of the baseline SHBGlevels. The low SHBG group had a mean SHBG baseline level of 44 nmol/L(±11), while the high SHBG group had a mean level of 183 nmol/L (±141).

Total Testosterone

In subjects with low SHBG, the three doses produced maximum levels oftotal testosterone of 3.18, 3.93 and 4.73 ng/mL, respectively, at 20 minafter dosing. In subjects with high SHBG, the maximum levels of totaltestosterone were 5.00, 7.08 and 9.04 ng/mL after administration of thethree doses sublingual testosterone. Between groups, total testosteronelevels were statistically different for t=10 till t=30 min in the 0.25and 0.50 mg dose, and in the 0.75 mg dose 6 till 30 min after dosing.

Free Testosterone

In subjects with low SHBG, the three doses produced maximum levels offree testosterone of 0.026, 0.039 and 0.048 ng/mL, respectively, at 20min after dosing. In subjects with high SHBG, the maximum levels of freetestosterone were 0.018, 0.026 and 0.034 ng/mL after administration ofthe three doses sublingual testosterone. Between groups, all differenceswere statistically different, except for the levels of free testosteronein the 0.25 mg dose 4 and 20 min after dosing and in the 0.75 mg dose 4and 10 min after dosing.

Our analyses showed that the low SHBG group had overall significantlyhigher levels of the free fraction compared to the high SHBG group(P=0.007). Analyses revealed a statistically significant Group×Drugeffect for the difference between 0.25 mg and 0.75 mg (P=0.012) andbetween 0.25 mg and 0.50 mg (P=0.031) (see FIG. 9). As shown in FIG. 9,statistically significant differences between the different dosessublingual testosterone were found in the low SHBG group.

Secondary Pharmacokinetic Endpoints

DHT peak levels of 0.285, 0.404 and 0.465 ng/mL were reached at means of27.5, 28.0 and 27.5 min respectively (Table 10).

The max differences between the three doses were not significant. Thedifference between the Cmax of the 0.25 mg vs. 0.50 mg and 0.25 mg vs.0.75 mg was significant (P<0.0001), and the difference between the Cmaxof 0.50 mg and 0.75 mg was statistically significant (P=0.0310). Meanresidence time of were not different the three sublingual doses. AUCswere statistically significant different between the three doses andincreased dose-dependently.

The difference between the AUCs of the 0.25 mg vs 0.50 mg and 0.25 mg vs0.75 mg was statistically significant (P<0.0001), while the differencebetween the 0.50 and 0.75 mg was significant at P=0.0208. There were nostatistically significant differences between the three doses, for thecalculated half-life of DHT. For all doses, return to DHT baselinelevels occurred within 180 min (FIG. 10).

Increasing doses of sublingual testosterone does not seem to influencethe 3α-diol-G concentrations as measured at t=0, t=60, t=120, and t=230.Cmax and AUCs differences were not statistically significant between thethree doses. E₂ levels did not change between the three doses ofsublingual testosterone and did not increase significantly compared tobaseline on t=60 and t=230 min (data not shown).

The three doses sublingual testosterone were well tolerated.

DISCUSSION

Our results demonstrate that sublingual administration of each of thethree doses testosterone was followed by a quick and steep increase oftotal and free testosterone levels; with peak levels reached at 15 min.Serum levels of total and free testosterone rapidly declined to reachbaseline levels by 2.5 h, which is in line with our previous study(Davison et al., 2005; Tuiten et al., 2000), and with the reportedpharmacokinetic profile following inhalation of testosterone (Davison etal., 2005).

The total testosterone Cmax following administration of 0.50 mgsublingual testosterone showed consistency with the reported Cmax ofTuiten et al (Tuiten et al., 2000). Also, the time to reach Cmax oftotal testosterone in this study showed uniformity with the data ofTuiten et al. and the study of Salehian et al., who administered 2.5 mgand 5.0 mg sublingual testosterone (Salehian et al., 1995).

DHT levels showed a significant dose-dependent increase, peak levelswere reached within 30 min and levels returned to baseline levels within3 h. DHT is metabolized to 3α-diol-G, so an elevation of 3α-diol-Glevels was expected after administration of sublingual testosterone.However, no dose-dependent effect of sublingual testosterone on theconcentration of 3α-diol-G was found.

According to the SHBG saturation threshold hypothesis by van der Made etal. (van der Made et al., 2009), an increased influx of testosteroneinto the body will occupy binding sites of SHBG. When most binding sitesare occupied, free (non-SHBG bound) testosterone and consequently thefree fraction will increase and thereby inducing, probably via genomicmechanisms (Bos et al., 2011), behavioral effects after approximately 4h.

The results of the present study show that free and total testosteronelevels significantly increase dose-dependently, which is reflected by anincrease in the free fraction of testosterone. However, the differencein free fraction of testosterone between the 0.50 and 0.75 mg conditiondid not reach statistical significance. It is interesting that aroundTmax of free and total testosterone, six women have lower free fractionlevels in the 0.75 mg condition compared to the 0.50 mg condition.Whether this is the result of variation in drug absorption, or the largebetween-subject variation in SHBG levels which could have influenced theresults, is not clear. Furthermore, it is also possible that the numberof subjects was probably too small to detect a significant increase infree fraction levels between these two doses.

Testosterone has a high affinity to SHBG and slowly dissociates fromSHBG. Free testosterone is rapidly metabolized (T½ 10 min.) whichdemonstrates the importance of SHBG binding and dissociation capacity,indicating that SHBG is the major determinant of the free fractionequilibrium. FIG. 4 shows the free fraction levels for subjects with lowand high SHBG levels. In the low SHBG group we observed an increase ofthe free fraction of testosterone levels induced by increasing dosagesof sublingual testosterone, while this pattern was not found in thewomen with high SHBG. These results corroborate the hypothesis of vander Made et al. (van der Made et al., 2009), namely: absorbedtestosterone is bound to SHBG which has a limited capacity and only whenthis binding capacity is saturated, free testosterone and the freefraction increase.

According to van der Made, the increase in the free fraction might beresponsible for behavioral effects observed 3.5 to 4 h later. However,in this study we measured free testosterone levels directly (withLC/MSMS) and we found these to be dose-dependently increased in bothSHBG groups, in contrast to the free fraction which did not show adose-dependent increase. Therefore we propose an adjustment to the SHBGsaturation threshold hypothesis as postulated by van der Made et al (vander Made et al., 2009); it is confirmed that SHBG levels influence thepercentage of free fraction of testosterone (and the maximumconcentration of free testosterone), however, an increase in freetestosterone levels seems to be relatively less dependent of circulatingSHBG levels after administration of the used dosages of sublingualtestosterone. Further studies are necessary to investigate if freetestosterone levels or free fraction levels are responsible to theobserved behavioral effects as described by van der Made et al.

The data of the bioavailability show that sublingual testosteroneabsorption decreases with increasing doses and is 69% and 58% for the0.50 and 0.75 dose respectively when the 0.25 mg condition is used asthe reference value (100%). These data suggest a limitation of the totalamount of testosterone absorbed. The volumes of the sublingualtestosterone solution in the higher dose conditions were larger comparedto the lower dosages. These increasing volumes could possibly influencethe absorption at the limited surface area in the mouth.

In this study we did not take into account the cyclical and diurnalvariation of testosterone. It is well known that testosterone levels arehighest during the ovulatory and midluteal phase of the menstrual cycleand lowest in the early follicular phase and late luteal phase (Judd andYen, 1973; Rothman et al., 2011; Salonia et al., 2008). In this study,blood samples were taken irrespective of menstrual cycle phase. However,almost 60% of the women in this study used some form of hormonalcontraceptive (combined oral contraceptive pill, combined-contraceptivevaginal ring) which is known to suppress ovulation (Bancroft et al.,1991; Mulders and Dieben, 2001). Moreover, we assumed that the useddosages used in the present study overruled considerably the naturaloccurring relatively subtle cyclical and diurnal variation oftestosterone. Furthermore, in a recent study by Braunstein et al. it wasshown that SHBG levels of 161 women remained relatively stable acrossthe menstrual cycle. They found a relatively small increase intestosterone levels in the mid-cycle period compared to the overallvariability and suggest that the reference ranges described can beapplied irrespective of the day in the menstrual cycle (Braunstein etal., 2011). So it is therefore unlikely that the dose-dependent increasein total and free testosterone levels are biased by the cyclical anddiurnal variation of testosterone.

Next to the sublingual route of testosterone administration other routescould be investigated as well. However for the desired immediate uptakeand rapid return of testosterone to baseline levels the intramuscularand transdermal route are not suitable since both will result in gradualsystemic uptake and prolonged higher plasma levels after drugadministration via these routes. Oral administration is impossible atall, since due to the very large first-pass effect no unmodifiedtestosterone will reach the systemic circulation. For alternative routesnext to sublingual with a very fast uptake and quick return to baselineof testosterone, the pulmonal and nasal delivery could perhaps be usedfor which in that case suitable and convenient dosage forms need to bedeveloped.

In conclusion, the three doses testosterone are rapidly absorbed by thesublingual route and quickly metabolized without sustained elevations ofDHT and E₂. These data suggest that a SHBG threshold exists whichinfluences the increase in free fraction levels.

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TABLE 7 Function Weight in mg. Coated Inner Sildenafil Core Sildenafilcitrate Active DMF 70.24 Dicalcium phosphate anhydrous Filler USP 102.88Microcrystalline cellulose (Avicel PH200) Filler USP/NF 102.88Croscarmellose sodium Disintegrant USP/NF 12.00 Magnesium stearateLubricant USP/NF 12.00 Ethylcellulose 20 cps^(b) Barrier coating USP/NF14.00 Microcrystalline cellulose (Avicel PH105)^(b) Coating pore formerUSP/NF 28.00 Subtotal: 342.00 Outer Testosterone Coating TestosteroneActive USP 0.5 Hypromellose 5 cps Coating polymer USP 1.34 Hydroxypropylβ-cyclodextrin Solubilizer USP/NF 2.66 Aspartame Sweetener USP/NF 1.00Menthol Flavor USP 0.60 Subtotal: 6.1 Total: 348.1

TABLE 8 Characteristic Value (n = 16) Age_yr 27.3 ± 5.3 Race_no (%)caucasian 11 (69) black  2 (13) asian  1 (6) other  2 (13)^(a) BMI_kg/m²23.5 ± 3.4 Contraceptive_no (%) hormonal 11 (69) combined oralcontraceptive pill  8 (50) IUD (levonorgestrel)  2 (13) vaginal ring(progestin and estrogen)  1 (6) non-hormonal  1 (6) none  4 (25.0) Totaltestosterone_ng/mL  0.2 ± 0.1 Free testosterone_pg/mL  1.9 ± 0.7^(b)DHT_ng/mL  0.1 ± 0.03 3α-diol-G_ng/mL  2.0 ± 1.9 E₂_pmol/L  207 ±147^(c) SHBG_nmol/L  114 ± 120 Albumin_g/L 44.7 ± 1.5 Plus-minus valuesare means ±SD. To convert total testosterone to nanomoles per liter,multiply by 3.467; to convert free testosterone to picomoles per liter,multiply by 3467. To convert total DHT to nanomoles per liter, multiplyby 3.44. To convert 3α-diol-G to nanomoles per liter, multiply by 2.13.All baseline levels are means of placebo, 0.25, 0.50, 0.75 mg predoselevels. ^(a)The percentages do not sum up to 100% due to rounding of thenumbers. ^(b)Only measured in 11 subjects; 5 subjects had values belowthe LLOQ. ^(c)Only measured in 15 subjects; 1 subject had a value belowthe LLOQ.

TABLE 9 Baseline corrected AUC Dose t_(1/2) * T_(max) * 0-230 ** C_(max)** MRT * (mg) (min) (min) (ng * min/mL) (ng/mL) (min) Testosterone 0.2549.8 ± 16.0 15.6 ± 5.4 194 (37.2)  3.79 (39.9) 57.7 ± 12.2 (ng/mL) ^(a)0.50 49.7 ± 22.4 15.1 ± 5.5 266 (37.6)  5.31 (37.8) 55.6 ± 13.9 0.7558.5 ± 24.6 14.3 ± 5.3 337 (34.7)  6.73 (39.6) 59.5 ± 16.4 Free 0.2542.3 ± 14.6 15.6 ± 5.1 0.95 (51.8) 0.021 (39.7) 52.6 ± 11.6 testosterone0.50 55.7 ± 27.5 14.4 ± 5.5 1.51 (40.2) 0.032 (37.6) 57.1 ± 15.6 (ng/mL)^(b) 0.75 51.1 ± 26.4 12.8 ± 6.3 1.87 (47.8) 0.043 (45.7) 51.4 ± 14.5^(a) Total testosterone normal range = 0.14 to 0.66 ng/mL (Davison etal., 2005). ^(b) Free testosterone normal range = 0.00072 to 0.0036ng/mL (Davison et al., 2005). To convert total testosterone to nanomolesper liter, multiply by 3.467; to convert free testosterone to picomolesper liter, multiply by 3467. MRT = mean residence time * mean ± SD **geometric mean (% CV)

TABLE 10 Dose t_(1/2) * T_(max) * AUC 0-230 C_(max) ** MRT * (mg) (min)(min) (ng * min/mL) (ng/mL) (min) Dihydro- 0.25 45.1 ± 10.5 27.5 ± 4.520.6 (44.9) 0.285 (42.5) 75.7 ± 14.4 testosterone 0.50 44.5 ± 16.8 28.0± 4.1 28.8 (37.9) 0.404 (37.6) 73.4 ± 14.8 (ng/mL) 0.75 50.5 ± 30.4 27.5± 4.5 34.4 (41.3) 0.465 (43.5) 81.5 ± 36.3 DHT reference range =<0.29ng/mL (Davison et al., 2005) To convert total DHT to nanomoles perliter, multiply by 3.44. * mean ± SD ** geometric mean (% CV)

Example 7 Development of Buspirone Core Formulation

The formulation of a Buspirone core was based on the Sildenafil 50 mgcore. The same excipients were used for development of a BuspironeHydrochloride core and a similar “direct compression” manufacturingprocess. The formulation combines a water insoluble filler (DicalciumPhosphate Anhydrous) with a water insoluble binder (MicrocrystallineCellulose) and a small amount of a super-disintegrant (CroscarmelloseSodium). This formulation is designed to give consistent stressrelaxation of the core and rupture of the barrier coat (after wateringress through the barrier coat), and rapid release of the BuspironeHydrochloride (after coat rupture).

A “direct compression” manufacturing process was used and directcompression grades of Dicalcium Phosphate Anhydrous (A-Tab, manufacturedby Innophos) and Microcrystalline Cellulose (Avicel PH-200, manufacturedby FMC Biopolymer) were selected to provide good flow properties and theability to form hard tablets.

Formulation of Buspirone Hydrochloride 10 mg Cores

Amount (mg per Amount Item Material tab) (%) Function 1. BuspironeHydrochloride 10.0 3.08 Active 2. Microcrystalline cellulose 97.5 30.00Filler/binder (Avicel PH-200) 3. Dicalcium phosphate anhydrous 200.161.57 Filler (A-TAB) 4. Croscarmellose sodium 13.0 4.00 Disintegrant(Ac-Di-Sol) 5. Magnesium stearate 4.4 1.35 Lubricant (vegetable source)Total 325.0 100.0

Cores made using this formulation and blending process had good physicalproperties, good content uniformity and disintegrated rapidly (in lessthan 1 minute), giving complete dissolution of Buspirone Hydrochloridein 15 minutes (using USP Apparatus 3, 250 ml of pH 4.5 sodium acetatebuffer and 20 dips per minute). Test results are summarised in Tables11-14 below.

TABLE 11 Physical Properties of Buspirone Hydrochloride 10 mg Cores Coreproperty Test results Friability (100 revolutions) 0.14% Friability (375revolutions) 0.33% Disintegration time range (6 cores) 18-25 seconds(Results for Batch No. 2112/46)

TABLE 12 Buspirone Hydrochloride Dissolution from 10 mg Uncoated CoresTime % Dissolved (6 tablets) (minutes) Average Range 15  98 97-99  30100 99-101 45 100 99-101 60 101 99-102 Test method = USP Apparatus 3,250ml of pH 4.5 sodium acetate buffer, 20 dips per minute. Results forBatch No. 2112/46

Development of Barrier Coating for Buspirone Cores

A barrier coating formulation and process have been developed in aperforated pan coater. The coating is designed to release the API 120 to180 minutes after the start of in-vitro dissolution testing. A waterinsoluble coating (ethylcellulose 20 cps [Ethocel 20]) was combined withmicrocrystalline cellulose [Avicel PH-105]), to allow controlled wateringress to cause gradual stress relaxation of the inner core andeventually cause rupturing of the insoluble coating in a pH independentmanner.

The same coating suspension and coating process were used for BuspironeHydrochloride cores as for sildenafil cores.

TABLE 13 Formulation of barrier coating suspension Material AmountFunction Ethylcellulose 20 cps  30.0 g Water insoluble coating polymer(Ethocel 20) Microcrystalline cellulose  60.0 g Membrane regulationagent (Avicel PH-105) Ethanol 96%  1000 ml Solvent

An experimental pan load of Buspirone Hydrochloride 10 mg cores wascoated to determine the amount of barrier coating required to give adelayed release of between 120 and 180 minutes, and to determine theeffect of a heat treatment (curing) step after applying the barriercoat.

Selected samples were dried in a lab oven for 15 hours at 60 deg C. andretested, to determine the effect of heat treatment. The results aresummarized in table 14.

TABLE 14 Rupture times of samples of Buspirone Hydrochloride 10 mgbarrier coated tablets, before and after heat treatment in a lab ovenSpraying time (minutes) 120 135 150 165 Weight of suspension sprayed (g)1191 1339 1487 1638 Average coat weight (mg/tab) 34.9 39.4 43.3 48.4 a)Rupture time of samples tested before heat treatment (n = 6): Average(minutes) 75.0 102.3 123.7 155.2 Range (minutes) 66-81 84-127 107-133142- 197 SD (minutes) 4.9 16.2 9.9 20.8 b) Rupture time of samplestested after heat treatment (n = 6): Average (minutes) Not 128.0 142.2Not tested tested Range (minutes) 92-188 118-162 SD (minutes) 32.3 15.6Batch No. 2112/56 Heat treatment = 15 hours at 60 deg C. in lab oven.

The results show that a coat weight of approximately 44 mg is requiredto achieve rupture times of between 120 and 180 minutes, after heattreatment, and that the heat treatment step increases the averagerupture time by about 20 minutes.

A further pan load of Buspirone Hydrochloride 10 mg cores was barriercoated to investigate heat treatment in the coating pan.

TABLE 15 Rupture times of Buspirone Hydrochloride 10 mg barrier coatedtablets, before and after heat treatment in the coating pan Sprayingtime (minutes) 140 154 154 154 Weight of suspension sprayed (g) 14001525 1525 1525 Average coat weight (mg/tab) 40.6 43.7 — — Heat treatmenttime (minutes) 0 0 60 90 Rupture time (n = 6): Average (minutes) 100.0135.3 149.2 145.4 # Range (minutes) 77-116 125-157 132-159 116-175 # SD(minutes) 15.9 13.1 9.7 15.6 # #12 tablets tested Batch No. 2112/60

The results were similar to the initial coating trial, indicating thatapproximately 44 mg of coating is required to achieve the target rupturetime of 120 to 180 minutes, combined with a heat treatment of 60 minutesin the coating pan. Heating for 90 minutes produces no significantchange in average rupture time, indicating that the “curing” process iscomplete after 60 minutes.

To summarize, a barrier coat weight of between 35 mg and 50 mg per core,preferably about 44 mg per core, was found to be required to give therequired time delay before rupture of the Buspirone Hydrochloride cores.A heating (curing) step seems to be required to stabilise the coating,to prevent changes in rupture time when coated tablets are stored. Theheating (curing) step was found to add about 20-30 minutes to theaverage rupture time of the tablets (comparing coated tablets before andafter the heat treatment).

Example 8 Clinical Study

A randomized, cross-over controlled study to compare the pharmacokineticprofiles of two combination products, a sublingual solution with anencapsulated tablet versus a combination tablet containing bothtestosterone and sildenafil citrate in healthy pre-menopausal women. Atotal of 12 subjects received in random order formulation 1 (F1):Testosterone (0.5 mg) administered sublingually as a solution, followed2.5 hours later by an encapsulated tablet containing 50 mg sildenafil assildenafil citrate or formulation 2 (F2): a fixed combination, tabletconsisting of an inner core component of 50 mg sildenafil, as sildenafilcitrate, coated with a polymeric coating designed to release thesildenafil citrate 2.5 hours after tablet intake. The coated sildenafilcore tablet is film-coated with an additional, immediately dissolving,polymeric, testosterone coating that releases 0.5 mg testosteronesublingually within 2 minutes.

The-first objective of this study was to compare the pharmacokinetics ofsublingual testosterone cyclodextrin followed by sildenafil citrate asan encapsulated tablet (F1) with administration of testosterone andsildenafil citrate as one tablet designed to release the components in aspecific time frame (F2).

The secondary objective was to investigate the time frame in which thetestosterone coating of the combination tablet is dissolvedsublingually.

Materials and Methods

EDTA whole blood samples of 12 subjects, receiving drug doses offormulation 1 (F1) and formulation 2 (F2) in random order, were taken atpre-dose (−10 min) and at 5, 10, 15, 20, 25, 30, 60, 90, 120, 135, 145,165, 180, 195, 210, 225. 240, 270, 300, 330, 360, 390, 450, 570, 690,810, 930 and 1590 minutes post-dose.

Blood samples, for the analysis of testosterone (T), free-testosterone(FT) and dihydro-testosterone (DHT) were taken at pre-dose and at 5, 10,15, 20, 25, 30, 60, 90, 120, 145, 160, 240 and 1590 minutes post-dose(total 14 time points).

Testosterone, dihydro-testosterone and free testosterone concentrationswere determined as described in Example 6.

Blood samples, for the analysis of sildenafil (S) andN-desmethyl-sildenafil (NDS) were taken for F1 at 145, 165, 180, 195,210, 225, 240, 270, 300, 330, 360, 390, 450, 570, 690, 810, 930 and 1590minutes post-dose (total 18 time points) and for F2 at pre-dose and at10, 30, 60, 90, 120, 135, 145, 165, 180, 195, 210, 225, 240, 270, 300,330, 360, 390, 450, 570, 690, 810, 930 and 1590 minutes post-dose (total25 time points).

Sildenafil (S) and N-desmethyl-sildenafil (NDS) concentrations weredetermined by HPLC-MS/MS as follows.

The human plasma samples were vortex mixed and 0.5 mL of the sample wastransferred into a clean test tube to which 20 μL of an InternalStandard solution (10 ng/mL) in methanol was added and vortex mixed.Then, 4 mL Methyl-Tertiary-Butyl-Ether (MTBE) was added, tubes werecapped and shaken for 10 minutes and then centrifuged for 5 minutes at2000 rcf. The tubes were placed into a snap freezer and the bottom waterlayer was frozen. The supernatant was transferred into a clean tube andevaporated to dryness under a stream of nitrogen. The residue wasreconstituted with 200 μL reconstitution solvent (50/50: MeOH/H₂Ocontaining 0.1% acetic acid), transferred to glass auto sampler vialsand arranged on the auto sampler tray. Injections of 7 μL were made forHPLC-MS/MS analysis.

The HPLC-MS/MS assay was carried out using the following equipment:

-   -   Analytical system: Applied Biosystem/MDS SCIEX API-4000 triple        quadrupole mass spectrometer with Analyst software    -   Mode: Positive Multiple Reaction Monitoring    -   Interface: Ion spray (Turbo spray)    -   HPLC-System: Shimadzu Co-sense system    -   HPLC column: Phenomenex Kinetex, C18 dimension 100×2.1 mm,        particle size 2.6 μm

Measurements (M/z): Sildenafil 475/283 N-desmethyl-sildenafil 461/283D₈-N-desmethyl-sildenafil 469/283

Pharmacokinetic Analysis

The software used for the pharmacokinetic analysis was Watson 7.2Bioanalytical LIMS software (Thermo ElectronCorporation-Philadelphia-USA).

Cmax and Tmax were read from the observed values. The half life wascalculated from the unweighted linear regression of the log transformeddata determined at the elimination phase of the pharmacokinetic profile.The Area Under Curve (O-last) was determined as the area under theconcentration versus time curve from the first time point to last timepoint with measurable drug concentration with a linear/log-lineartrapezoidal model. The AUC (0-∞) was determined by extrapolation fromthe time point where the last measurable drug concentration (Cp)occurred to time infinity. This was performed by dividing the observedconcentration at the last time point by the elimination rate constantdetermined using linear regression of Cp versus time data (standardextrapolation technique). Tlag was determined as the first time pointwith a measurable concentration.

Results

A total of 12 subjects received in random order both formulation 1 (F1)and formulation 2 (F2).

TABLE 16 Pharmacokinetic parameters of testosterone (T),free-testosterone (FT) and dihydro- testosterone (DHT), sildenafil (S)and N-desmethyl-sildenafil (NDS). Pharmacokinetic parameters forTestosterone AUC (0-last) Rate Constant (Az) Dosing Cmax (ng/mL) Tmax(hours) (ng*hours/mL) T1/2 (hours) (1/Hours) F1 5.66 ± 1.82 0.229 ±0.063 5.13 ± 1.08 0.615 ± 0.107 1.16 ± 0.207 F2 8.06 ± 2.07 0.205 ±0.065 7.69 ± 2.49 0.629 ± 0.088 1.12 ± 0.167 Pharmacokinetic parametersfor Free-testosterone AUC (0-last) Rate Constant (Az) Dosing Cmax(ng/mL) Tmax (hours) (ng*hours/mL) T1/2 (hours) (1/hours) F1 0.0318 ±0.0117 0.250 ± 0.0645 0.0276 ± 0.0167 0.652 ± 0.196 1.16 ± 0.380 F20.0455 ± 0.0181 0.242 ± 0.0693 0.0449 ± 0.0216 0.593 ± 0.109 1.21 ±0.239 Pharmacokinetic parameters for Dihydro-testosterone AUC (0-last)Rate Constant (Az) Dosing Cmax (ng/mL) Tmax (hours) (ng*hours/mL) T1/2(hours) (1/hours) F1 0.492 ± 0.169 0.438 ± 0.0722 1.07 ± 0.488 1.80 ±1.00 0.504 ± 0.273 F2 0.645 ± 0.232 0.485 ± 0.0337 1.22 ± 0.568  1.40 ±0.841 0.676 ± 0.366 Pharmacokinetic parameters for Sildenafil Cmax TmaxAUC (0-last) AUC Extrap (0-inf) Tlag T1/2 Rate Constant (Az) Dosing(ng/mL) (hours) (ng*hours/mL) (ng*hours/mL) (hours) (hours) (1/hours) F1268 ± 141 3.88 ± 1.08 577 ± 204 596 ± 203 3.23 ± 0.494 3.87 ± 2.04 0.217± 0.0856 F2 173 ± 82.7 3.10 ± 0.642 476 ± 133 500 ± 136 2.74 ± 0.6164.69 ± 2.02 0.175 ± 0.0722 Pharmacokinetic parameters forN-desmethyl-sildenafil Cmax Tmax AUC (0-last) AUC Extrap (0-inf) T1/2Rate Constant (Az) Dosing (ng/mL) (hours) (ng*hours/mL) (ng*hours/mL)Tlag (hours) (hours) (1/hours) F1 55.5 ± 20.2 4.00 ± 1.28 194 ± 90.6 203± 92.4 3.29 ± 0.620 5.21 ± 1.16 0.144 ± 0.0599 F2 42.7 ± 18.3 3.34 ±0.789 155 ± 50.2 171 ± 55.6 2.78 ± 0.717 7.07 ± 2.26 0.113 ± 0.0568

The mean concentrations of testosterone and free-testosterone from theplasma-time profiles measured after oral administration of a single doseof testosterone (0.5 mg) using the F1 and F2 dosing regime in healthypre-menopausal female subjects are shown in FIGS. 12 and 13.

The mean concentration of sildenafil from the plasma-time profilesmeasured after oral administration of a single dose of sildenafil (50mg) using the F1 and F2 dosing regimes in healthy pre-menopausal femalesubjects is shown in FIG. 14. Since testosterone is endogenous inplasma, for all calculations the predose concentration was subtractedfrom the determined concentration after dosing. The calculatedconcentrations were used for PK calculations. One subject was excludedfrom PK calculations for the F2 dosing group with the analysis oftestosterone, dihydro-testosterone and free-testosterone.

A further subject was not included in the free-testosterone PKcalculations for the F1 dosing group.

The pharmacokinetic results show that testosterone was rapidly absorbedwith a Tmax in the range between 10 and 20 minutes and an average halflife of approximately 37 minutes. Free-testosterone results showed apicture comparable to the testosterone results. Tmax and half life fordihydro-testosterone were however later than for testosterone. It isnoted that the average AUC with F2 dosing was higher for testosterone,dihydrotestosterone and free-testosterone compared to the F1 dosing.

Sildenafil exposure was prolonged and did not start until approximatelythree hours after first dosing. The average Tmax for sildenanil wasalmost 4 hours with F1 dosing and just over 3 hours with F2 dosing.N-desmethyl-sildenafil followed the same pattern as sildenafil, i.e. aTmax of just a few minutes later and a comparable half life. It is notedthat the average AUC with F1 dosing is higher for sildenafil andN-desmethyl-sildenafil compared to the F2 dosing.

The Tmax−Tlag for sildenafil using the F2 dosing is 3.10-2.74=0.36 h(see Table 16), which indicates that the maximal concentration ofsildenafil is reached very fast after the burst of the core of the dualdrug delivery device.

Example 9

Cores with a composition as shown in Table 17 were coated with 21.5 mgof ethylcellulose/avicel (1:1 w/w) coating. In vitro dissolution testsexperiments were carried out using a USP dissolution apparatus no. II(Prolabo, Rowa techniek BV) with a rotational speed of 50 rpm and 1000ml of medium at 37° C. (n=6). The dissolution media used was a citratebuffer, pH 4.5. The amount of sildenafil dissolved was determinedcontinuously by UV absorbance at a wavelength of 291 nm.

Representative examples of dissolution of individual tablets aredepicted in FIG. 15.

TABLE 17 Composition of cores Amount Material (mg per tablet) SildenafilCitrate 70.24 Microcrystalline cellulose (Avicel PH-200) 102.88Dicalcium phosphate anhydrous (A-TAB) 102.88 Croscarmellose sodium(Ac-Di-Sol) 12.0 Magnesium stearate (vegetable source) 12.0 Total 300.0

Example 10

Representative examples of dissolution experiments of individual tabletswith coated cores having a composition as shown in Table 18, aredepicted in FIG. 16.

In vitro dissolution tests experiments were carried out using a USPdissolution apparatus no. II (Prolabo, Rowa techniek BV) with arotational speed of 50 rpm and 1000 ml of medium at 37° C. (n=6). Thedissolution media used was a citrate buffer, pH 4.5. The amount ofsildenafil dissolved was determined continuously by UV absorbance at awavelength of 291 nm.

TABLE 18 Composition of coated cores Sildenafil citrate 70.24 Dicalciumphosphate anhydrous 102.88 Microcrystalline cellulose (Avicel PH200)102.88 Croscarmellose sodium 12.00 Magnesium stearate 12.00Ethylcellulose 20 cps 14.00 Microcrystalline cellulose (Avicel PH105)28.00 Subtotal: 342.00

1. A tablet for sublingual administration of a first active ingredientsaid tablet comprising a core, and an outer coating on the exteriorsurface of said tablet and optionally a separation coating thatseparates said outer coating from said core, wherein said outer coatingcomprises a mixture of said first active ingredient in amorphous form inan amount of between about 0.1-10 mg; a coating polymer in an amount ofbetween about 0.25-25 mg; and water in an amount of between about0.0-10% w/w of the outer coating.
 2. The tablet of claim 1, wherein thefirst active ingredient is testosterone or a functional analog thereof.3. The tablet of claim 1, wherein said mixture further comprises acyclodextrin or a polyvinylpyrolidone, or a combination thereof in anamount of between 0.25-25 mg.
 4. The tablet of claim 3, wherein saidmixture further comprises a cyclodextrin or a polyvinylpyrolidone, or acombination thereof in an amount of between 0.5-12.5 mg.
 5. The tabletof claim 1, wherein said mixture comprises said first active ingredientin an amount of between about 0.2-5.0 mg; said coating polymer in anamount of between about 0.5-12.5 mg; and water in an amount of betweenabout 0.0-5% w/w of the outer coating.
 6. The tablet of claim 1, whereinsaid mixture of said outer coating further comprises a sweetener and/ora flavor.
 7. The tablet of claim 1, wherein said mixture comprises 0.5mg of testosterone, 1.34 mg of hydroxypropyl methylcellulose, 2.66 mg ofhydroxypropyl beta-cyclodextrin, 1 mg of aspartame, and 0.6 mg ofmenthol.
 8. The tablet of claim 1, comprising said separation coatingthat separates said outer coating from said core.
 9. The tablet of claim8, wherein said core and said separation coating has a volume of between50-1000 mm³.
 10. The tablet of claim 8, wherein said core comprises acellulose, an inorganic salt and a second active ingredient.
 11. Thetablet of claim 8, wherein said separation coating comprises ahydrophobic polymer and a hydrophilic substance.
 12. The tablet of claim8, wherein said separation coating is a pH-independent coating.
 13. Thetablet of claim 12, wherein said separation coating is an acid solublecoating or an enteric coating.
 14. The tablet of claim 10, whichcomprises a time controlled, immediate release drug delivery system fororal administration of the second active ingredient to a subject in needthereof, the system comprising said core comprising cellulose, a fillerselected from an organic and/or an inorganic salt, and said secondactive ingredient, and said separation coating surrounding the corecomprising a hydrophobic polymer and a hydrophilic substance.
 15. Thetablet of claim 1, wherein said second active ingredient is selectedfrom the group consisting of a PDE5 inhibitor, a 5HT1A receptor agonist,and a neutral endopeptidase inhibitor.